Multivalent clostridial toxins

ABSTRACT

The present invention is directed to multivalent Clostridial toxin comprising more than one binding domain directed to a cell surface molecule of a target cell. Such modified toxins are useful as therapeutic compositions to prevent exocytosis and secretion by the target cell. Conditions in which such compositions man be useful include, without limitation, disorders of the sensory or motor nervous system, acute or chronic pain, cancer, pancreatitis, hyperhydrosis, glandular disorders, viral infections, cystic fibrosis and the like. The invention is also directed to methods of using and administering such a composition, and methods of treating a given condition using such a composition.

This is a continuation in part and claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 11/376,696, filed Mar. 15, 2006, now U.S. Pat. No. 7,514,088, a national stage application under 35 U.S.C. §371 of PCT application PCT/US 2005/020578, filed 9 June, 2005 which claims priority pursuant to 35 U.S.C. §119(e) to provisional patent application Ser. No. 60/584,378 filed Jun. 30, 2004 and provisional patent application Ser. No. 60/599,132 filed Aug. 4, 2004, which are all hereby incorporated by reference in their entirety.

The ability of Clostridial toxins to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic applications, see e.g., William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004). Clostridial toxins commercially available as pharmaceutical compositions include, BoNT/A preparations, such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.), Dysport®/Reloxin®, (Beaufour Ipsen, Porton Down, England), Linurase® (Prollenium, Inc., Ontario, Canada), Neuronox® (Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A (Lanzhou Institute Biological Products, China) and Xeomin® (Merz Pharmaceuticals, GmbH, Frankfurt, Germany); and BoNT/B preparations, such as, e.g., MyoBloc™/NeuroBloc™ (Elan Pharmaceuticals, San Francisco, Calif.). As an example, BOTOX® is currently approved in one or more countries for the following indications: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and VII nerve disorder.

Clostridial toxin therapies are successfully used for many indications. Generally, administration of a Clostridial toxin treatment is well tolerated. However, toxin administration in some applications can be challenging because of the larger doses required to achieve a beneficial effect. First, larger doses can increase the likelihood that the toxin may move through the interstitial fluids and the circulatory systems, such as, e.g., the cardiovascular system and the lymphatic system, of the body, resulting in the undesirable dispersal of the toxin to areas not targeted for toxin treatment. Such dispersal can lead to undesirable side effects, such as, e.g., inhibition of neurotransmitter release in neurons not targeted for treatment or paralysis of a muscle not targeted for treatment. For example, a patient administered a therapeutically effective amount of a BoNT/A treatment into the neck muscles for torticollis may develop dysphagia because of dispersal of the toxin into the oropharynx. Thus, there remains a need for improved Clostridial toxins that are effective at the site of treatment, but have negligible to minimal effects in areas not targeted for a toxin treatment.

Second, larger doses of a Clostridial toxin treatment may elicit an antibody response against the toxin. While a potent and effective treatment, the inhibition of neurotransmitter release and the resulting neuromuscular paralysis elicited by Clostridial toxin therapies is not permanent. The reversible nature of these paralytic effects requires periodic treatments in order to maintain the therapeutic benefits from this toxin. As a consequence of this repeated exposure, an immune response against a Clostridial toxin can occur in some patients which reduce or completely prevent the individual's responsiveness to further treatments, see, e.g., Joseph Jankovic, Botulinum toxin: Clinical Implications of Antigenicity and Immunoresistance, (SCIENTIFIC AND THERAPEUTIC ASPECTS OF BOTULINUM TOXIN, 409-415, Mitchell F. Brin et al., eds., Lippincott Williams & Wilkins, 2002); Dirk Dressler, Clinical Presentation and Management of Antibody-induced Failure of Botulinum Toxin Therapy, 19(Suppl. 8) MOV. DISORD. S92-S100 (2004); M. Zouhair Atassi, Basic Immunological Aspects of Botulinum Toxin Therapy, 19(Suppl. 8) MOV. DISORD. S68-S84, (2004). Thus, there remains a need for improved Clostridial toxins that maintain effective therapeutic benefits, but have reduced ability to evoke an immunogenic response against itself.

Moreover, a Clostridial toxin treatment inhibits neurotransmitter release by disrupting the exocytotic process used to secret the neurotransmitter into the synaptic cleft. However, it is believed that current Clostridial toxin therapies may by expanded to treat new indications beyond those diseases or disorders whose underlying pathophysiology is aberrant cholinergic motor neuron activity.

Thus, the growing clinical, therapeutic and cosmetic use of Clostridial toxins in therapies requiring larger doses necessitates the pharmaceutical industry to develop modified Clostridial toxins that are effective at the target site of the application, but reduce or prevent the undesirable side-effects associated with the dispersal of the toxins to unwanted locations and reduce or prevent an unwanted immunogenic response. Additionally, there is a great desire by the pharmaceutical industry to expand the use of Clostridial toxin therapies beyond its current myo-relaxant applications to treat sensory-based ailment, such as, e.g., various kinds of chronic pain, as well as non-neuronal based disorders, such as, e.g., pancreatitis. The present invention provides novel multivalent Clostridial toxins that greatly extended the number of therapeutic applications that can exploit the advantages offered by current Clostridial toxin therapies. These and related advantages are useful for various clinical, therapeutic and cosmetic applications, such as, e.g., the treatment of neuropathic disorders, eye disorders, pain, muscle injuries, headache, cardiovascular diseases, neuropsychiatric disorders, endocrine disorders, cancers, otic disorders, as well as, other disorders where administration of a multivalent Clostridial toxin to an individual can produce a beneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of neurotransmitter release and Clostridial toxin intoxication in a central and peripheral neuron. FIG. 1A shows a schematic for the neurotransmitter release mechanism of a central and peripheral neuron. The release process can be described as comprising two steps: 1) vesicle docking, where the vesicle-bound SNARE protein of a vesicle containing neurotransmitter molecules associates with the membrane-bound SNARE proteins located at the plasma membrane; and 2) neurotransmitter release, where the vesicle fuses with the plasma membrane and the neurotransmitter molecules are exocytosed. FIG. 1B shows a schematic of the intoxication mechanism for tetanus and botulinum toxin activity in a central and peripheral neuron. This intoxication process can be described as comprising four steps: 1) receptor binding, where a Clostridial toxin binds to a Clostridial receptor and initiates the intoxication process; 2) complex internalization, where after toxin binding, a vesicle containing the toxin/receptor complex is endocytosed into the cell; 3) light chain translocation, where multiple events result in the release of the active light chain into the cytoplasm; and 4) enzymatic target modification, where the active light chain of Clostridial toxin proteolytically cleaves its target SNARE substrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking and neurotransmitter release.

FIG. 2 shows the domain organization of naturally-occurring Clostridial toxins. The single chain form depicts the amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a H_(CN) translocation facilitating domain and a H_(CC) targeting domain. The di-chain loop region located between the translocation and enzymatic domains is depicted by the double SS bracket. This region comprises an endogenous di-chain loop protease cleavage site that upon proteolytic cleavage with a naturally-occurring protease, such as, e.g., an endogenous Clostridial toxin protease or a naturally-occurring protease produced in the environment, converts the single chain form of the toxin into the di-chain form. As depicted above the single-chain form, the H_(CC) targeting domain comprises the β-trefoil domain which comprises in an amino to carboxyl linear organization of an α-fold, a β4/β5 hairpin turn, a β-fold, a β8/β9 hairpin turn and a γ-fold.

FIG. 3 shows a ribbon diagram of BoNT/A illustrating the modular three-dimensional structure of the light chain (LC) comprising the enzymatic domain, the heavy chain H_(N) domain comprising the translocation domain, and the heavy chain H_(c) domain, including the heavy chain H_(CN) domain and the heavy chain H_(CC) domain, that comprises the binding domain.

FIG. 4 shows examples of domain arrangements of multivalent Clostridial toxins. FIG. 4A depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising a binding domain 1, a translocation domain, a binding domain 2 and an enzymatic domain, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the binding domain 2 and enzymatic domain. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the binding domain 1 and translocation domain, the translocation domain and binding domain 2, binding domain 2 and enzymatic domain, or any combination thereof. FIG. 4B depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, a binding domain 1, a translocation domain and a binding domain 2, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the enzymatic domain and binding domain 1. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the enzymatic domain and binding domain 1, the binding domain 1 and translocation domain, the translocation and binding domain 2, or any combination thereof. FIG. 4C depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 1 and a binding domain 2, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the enzymatic domain and translocation domain. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the enzymatic domain and translocation domain, the translocation domain and binding domain 1, the binding domain 1 and a binding domain 2, or any combination thereof. FIG. 4D depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 2 and a binding domain 1, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the enzymatic domain and translocation domain. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the enzymatic domain and translocation domain, the translocation domain and binding domain 2, the binding domain 2 and a binding domain 1, or any combination thereof.

FIG. 5 shows examples of domain arrangements of multivalent Clostridial toxins. FIG. 5A depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising a binding domain 1, an enzymatic domain, a translocation domain and a binding domain 2, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the enzymatic domain and translocation domain. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the binding domain 1 and the enzymatic domain, the enzymatic domain and translocation domain, the translocation domain and binding domain 2, or any combination thereof. FIG. 5B depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, a binding domain 1 and an enzymatic domain, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the binding domain 2 and binding domain 1. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the translocation domain and binding domain 2, the binding domain 2 and binding domain 1, the binding domain 1 and enzymatic domain, or any combination thereof. FIG. 5C depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an enzymatic domain and a binding domain 1, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the binding domain 2 and enzymatic domain. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the translocation domain and binding domain 2, the binding domain 2 and enzymatic domain, the enzymatic domain and binding domain 1, or any combination thereof. FIG. 5D depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising a binding domain 2, a translocation domain, a binding domain 1 and an enzymatic domain, with the di-chain loop region depicted by the double SS bracket. A proteolytic cleavage site (P) within a di-chain loop region is located between the translocation domain and binding domain 1. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Spacers can be placed between the binding domain 2 and translocation domain, the translocation domain and binding domain 1, the binding domain 1 and enzymatic domain, or any combination thereof.

FIG. 6 shows examples of domain arrangements of multivalent Clostridial toxins. FIG. 6A depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 1 and a binding domain 2, with the di-chain loop region depicted by the double SS bracket. A first protease cleavage site (P) within a di-chain loop region is located between the enzymatic domain and translocation domain. A second protease cleavage site (P) is located between the binding domain 1 and binding domain 2. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the tri-chain form. The P protease site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Additionally, the first and second proteolytic cleavage sites can be the same site cleaved by the same protease or different sites cleaved by different proteases. Spacers can be placed between the enzymatic domain and translocation domain, the translocation domain and binding domain 1, the binding domain 1 and binding domain 2, or any combination thereof. FIG. 6B depicts the single polypeptide form of a multivalent Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 2 and a binding domain 1, with the di-chain loop region depicted by the double SS bracket. A first protease cleavage site (P) within a di-chain loop region is located between the enzymatic domain and translocation domain. A second protease cleavage site (P) is located between the binding domain 2 and binding domain 1. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the tri-chain form. The P protease cleavage site can be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial toxin exogenous protease cleavage site. Additionally, the first and second protease cleavage sites can be the same site cleaved by the same protease or different sites cleaved by different proteases. Spacers can be placed between the enzymatic domain and translocation domain, the translocation domain and binding domain 2, the binding domain 2 and binding domain 1, or any combination thereof.

DETAILED DESCRIPTION

The present invention provides novel Clostridial toxins that greatly extended the number of therapeutic applications that can exploit the advantages offered by current Clostridial toxin therapies. These multivalent Clostridial toxins comprise, in part, multiple binding domains. Each binding domain can be capable of binding 1) the cognate Clostridial toxin receptor present on the surface of a naturally-occurring Clostridial toxin target cell; 2) a different receptor present on the surface of a naturally occurring Clostridial toxin target cell; or 3) a different receptor present on the surface of the non-Clostridial toxin target cell. As such, a multivalent Clostridial toxin comprising multiple binding domains can exhibit increased specificity, efficacy and efficiency by which such multivalent Clostridial toxins can interact with a particular target cell and enzymatically modify its target SNARE substrate. Additionally, a multivalent Clostridial toxin comprising multiple binding domains can exhibit increased the versatility and therapeutic scope of current Clostridial toxin therapeutic applications by simultaneously targeting multiple cell types responsible for different aspects of a diseased state, such as, e.g., a nerve spasticity symptom and a pain symptom, or an aberrant enzyme release symptom and a pain symptom.

Aspects of the present invention a composition comprising a multivalent Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a first binding domain and a second binding domain, wherein each of the binding domains is independently capable of binding a cell surface receptor of a target cell. It is envisioned that the first binding domain and second binding domain may be identical to each other or different.

Other aspects of the present invention provide polynucleotide molecules encoding a multivalent Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a first binding domain and a second binding domain.

Other aspects of the present invention provide methods of producing a multivalent Clostridial toxin disclosed in the present specification, the method comprising the step of expressing in a cell a polynucleotide molecule encoding a multivalent Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a first binding domain and a second binding domain. Other aspects of the present invention provide methods of producing a multivalent Clostridial toxin disclosed in the present specification, the method comprising the steps of introducing in a cell an expression construct comprising a polynucleotide molecule encoding a multivalent Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a first binding domain and a second binding domain and expressing the expression construct in the cell.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii and Clostridium butyricum are the most widely used in therapeutic and cosmetic treatments of humans and other mammals. Strains of C. botulinum produce seven antigenically-distinct types of Botulinum toxins (BoNTs), which have been identified by investigating botulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and /D), or isolated from soil (BoNT/G). BoNTs possess approximately 35% amino acid identity with each other and share the same functional domain organization and overall structural architecture. It is recognized by those of skill in the art that within each type of Clostridial toxin there can be subtypes that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, with specific subtypes showing approximately 89% amino acid identity when compared to another BoNT/A subtype. While all seven BoNT serotypes have similar structure and pharmacological properties, each also displays heterogeneous bacteriological characteristics. In contrast, tetanus toxin (TeNT) is produced by a uniform group of C. tetani. Two other species of Clostridia, C. baratii and C. butyricum, also produce toxins, BaNT and BuNT respectively, which are similar to BoNT/F and BoNT/E, respectively.

Clostridial toxins are each translated as a single chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease (FIG. 2). This cleavage occurs within the discrete di-chain loop region created between two cysteine residues that form a disulfide bridge. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by the single disulfide bond and non-covalent interactions between the two chains. The naturally-occurring protease used to convert the single chain molecule into the di-chain is currently not known. In some serotypes, such as, e.g., BoNT/A, the naturally-occurring protease is produced endogenously by the bacteria serotype and cleavage occurs within the cell before the toxin is release into the environment. However, in other serotypes, such as, e.g., BoNT/E, the bacterial strain appears not to produce an endogenous protease capable of converting the single chain form of the toxin into the di-chain form. In these situations, the toxin is released from the cell as a single-chain toxin which is subsequently converted into the di-chain form by a naturally-occurring protease found in the environment.

TABLE 1 Clostridial Toxin Reference Sequences and Regions H_(C) Toxin SEQ ID NO: LC H_(N) H_(CN) H_(CC) BoNT/A 1 M1-K448 A449-I873 I874-P1110 Y1111-L1296 BoNT/B 2 M1-K441 A442-I860 L861-E1097 Y1098-E1291 BoNT/C1 3 M1-K449 T450-I868 N869-E1111 Y1112-E1291 BoNT/D 4 M1-R445 D446-I864 N865-E1098 Y1099-E1276 BoNT/E 5 M1-R422 K423-I847 K848-E1085 Y1086-K1252 BoNT/F 6 M1-K439 A440-I866 K867-K1105 Y1106-E1274 BoNT/G 7 M1-K446 S447-I865 S866-Q1105 Y1106-E1297 TeNT 8 M1-A457 S458-L881 K882-E1127 Y1128-D1315 BaNT 9 M1-K431 N432-I857 I858-K1094 Y1095-E1268 BuNT 10 M1-R422 K423-I847 K848-E1085 Y1086-K1251

Each mature di-chain molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets core components of the neurotransmitter release apparatus; 2) a translocation domain contained within the amino-terminal half of the HC(H_(N)) that facilitates release of the LC from intracellular vesicles into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the HC (H_(C)) that determines the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell. The H_(C) domain comprises two distinct structural features of roughly equal size that indicate function and are designated the H_(CN) and H_(CC) subdomains. Table 1 gives approximate boundary regions for each domain and subdomain found in exemplary Clostridial toxins. When discussing the three general neurotoxin domains of each clostridial neurotoxin subtype (binding, translocation and endopeptidase) it will be understood that clostridial neurotoxin research is a well-developed field, and the correlation of the amino acid sequences comprising each of these domains with their functions is well known. Additionally, the nucleotide and amino acid sequences of each of these domains are known and have been disclosed in this specification.

The binding, translocation and enzymatic activity of these three functional domains are all necessary for toxicity. While all details of this process are not yet precisely known, the overall cellular intoxication mechanism whereby Clostridial toxins enter a neuron and inhibit neurotransmitter release is similar, regardless of type. Although the applicants have no wish to be limited by the following description, the intoxication mechanism can be described as comprising at least four steps: 1) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) enzymatic target modification (see FIG. 1). The process is initiated when the H_(C) domain of a Clostridial toxin binds to a toxin-specific receptor complex located on the plasma membrane surface of a target cell. The binding specificity of a receptor complex is thought to be achieved, in part, by specific combinations of gangliosides and protein receptors that appear to distinctly comprise each Clostridial toxin receptor complex. Once bound, the toxin/receptor complexes are internalized by endocytosis and the internalized vesicles are sorted to specific intracellular routes. The translocation step appears to be triggered by the acidification of the vesicle compartment. This process seems to initiate two important pH-dependent structural rearrangements that increase hydrophobicity and promote formation di-chain form of the toxin. Once activated, light chain endopeptidase of the toxin is released from the intracellular vesicle into the cytosol where it specifically targets one of three known core components of the neurotransmitter release apparatus. These core proteins, vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking and fusion at the nerve terminal and constitute members of the soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminal region, releasing a nine or twenty-six amino acid segment, respectively, and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conserved central portion of VAMP, and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the cytosolic membrane surface. The selective proteolysis of synaptic SNAREs accounts for the block of neurotransmitter release caused by Clostridial toxins in vivo. The SNARE protein targets of Clostridial toxins are common to exocytosis in a variety of non-neuronal types; in these cells, as in neurons, light chain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al., Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C) domain of TeNT indicate that the three functional domains of Clostridial neurotoxins are structurally distinct domains that are shared by all Clostridial toxins. The HEXXH consensus motif of the light chain forms the tetrahedral zinc binding pocket of the catalytic site located in a deep cleft on the protein surface that is accessible by a channel. The structure of the H_(N) and H_(C) domains consists primarily of β-sheet topologies that are linked by a single α-helix. The cylindrical-shaped H_(N) domain comprises two long amphipathic α-helices that resemble the coiled-coil motif found in some viral proteins. The H_(N) domain also forms a long unstructured loop called the ‘translocation belt,’ which wraps around a large negatively charged cleft of the light chain that blocks access of the zinc atom to the catalytic-binding pocket of active site. The H_(C) domain comprises two distinct structural features of roughly equal size that indicate function. The first, designated the H_(CN) domain, is located in the amino half of the H_(C) domain. The H_(CN) domain forms a β-barrel, jelly-roll fold. The H_(CC) domain is the second domain that comprises the H_(C) domain. This carboxyl-terminal domain comprises a modified β-trefoil domain which forms three distinct carbohydrate binding regions that resembles the carbohydrate binding moiety found in many sugar-binding proteins, such as, e.g., serum amyloid P, sialidase, cryia, insecticidal ∂-endotoxin and lectins. Biochemical studies indicate that the β-trefoil domain structure of the H_(CC) domain appears to mediate the binding to specific carbohydrate containing components of the Clostridial toxin receptor on the cell surface, see, e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family Provides Residue Level Information About the Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124 (2000). The H_(C) domain tilts away from the H_(N) domain exposing the surface loops and making them accessible for binding. No contacts occur between the light chain and the H_(C) domain.

Aspects of the present invention provide, in part, a Clostridial toxin. As used herein, the term “Clostridial toxin” means any neurotoxin produced by a Clostridial toxin strain that can execute the overall cellular mechanism whereby a Clostridial toxin intoxicates a cell and encompasses the binding of a Clostridial toxin to a low or high affinity receptor complex, the internalization of the toxin/receptor complex, the translocation of the Clostridial toxin light chain into the cytoplasm and the enzymatic modification of a Clostridial toxin substrate. Exemplary Clostridial toxins include those produced by a Clostridium botulinum, a Clostridium tetani, a Clostridium baratii and a Clostridium butyricum.

A Clostridial toxin includes, without limitation, naturally occurring Clostridial toxin variants, such as, e.g., Clostridial toxin isoforms and Clostridial toxin subtypes; non-naturally occurring Clostridial toxin variants, such as, e.g., conservative Clostridial toxin variants, non-conservative Clostridial toxin variants, Clostridial toxin chimeric variants and active Clostridial toxin fragments thereof, or any combination thereof. As used herein, the term “Clostridial toxin variant,” whether naturally-occurring or non-naturally-occurring, means a Clostridial toxin that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Table 1) and can be described in percent identity to the corresponding region of that reference sequence. As non-limiting examples, a BoNT/A variant comprising amino acids 1-1296 of SEQ ID NO: 1 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1296 of SEQ ID NO: 1; a BoNT/B variant comprising amino acids 1-1291 of SEQ ID NO: 2 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1291 of SEQ ID NO: 2; a BoNT/C1 variant comprising amino acids 1-1291 of SEQ ID NO: 3 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1291 of SEQ ID NO: 3; a BoNT/D variant comprising amino acids 1-1276 of SEQ ID NO: 4 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1276 of SEQ ID NO: 4; a BoNT/E variant comprising amino acids 1-1252 of SEQ ID NO: 5 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1252 of SEQ ID NO: 5; a BoNT/F variant comprising amino acids 1-1274 of SEQ ID NO: 6 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1274 of SEQ ID NO: 6; a BoNT/G variant comprising amino acids 1-1297 of SEQ ID NO: 7 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1297 of SEQ ID NO: 7; a TeNT variant comprising amino acids 1-1315 of SEQ ID NO: 8 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1315 of SEQ ID NO: 8; a BaNT variant comprising amino acids 1-1268 of SEQ ID NO: 9 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1268 of SEQ ID NO: 9; and a BuNT variant comprising amino acids 1-1251 of SEQ ID NO: 10 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1-1251 of SEQ ID NO: 10.

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

Hybrid methods combine functional aspects of both global and local alignment methods. Non-limiting methods include, e.g., segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al., Multiple DNA and Protein Sequence Alignment Based On Segment-To-Segment Comparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996); T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A Novel Algorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217 (2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple Sequence Alignment With High Score Accuracy and High Throughput, 32(5) Nucleic Acids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran R Subramanian et al., DIALIGN-T: An Improved Algorithm for Segment-Based Multiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

As used herein, the term “naturally occurring Clostridial toxin variant” means any Clostridial toxin produced without the aid of any human manipulation, including, without limitation, Clostridial toxin isoforms produced from alternatively-spliced transcripts, Clostridial toxin isoforms produced by spontaneous mutation and Clostridial toxin subtypes. Non-limiting examples of a Clostridial toxin isoform include, e.g., BoNT/A isoforms, BoNT/B isoforms, BoNT/C1 isoforms, BoNT/D isoforms, BoNT/E isoforms, BoNT/F isoforms, BoNT/G isoforms, TeNT isoforms, BaNT isoforms and BuNT isoforms. Non-limiting examples of a Clostridial toxin subtype include, e.g., BoNT/A subtypes BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4; BoNT/B subtypes BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B nonproteolytic; BoNT/C1 subtypes BoNT/C1-1 and BoNT/C1-2; BoNT/E subtypes BoNT/E1, BoNT/E2 and BoNT/E3; and BoNT/F subtypes BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4.

As used herein, the term “non-naturally occurring Clostridial toxin variant” means any Clostridial toxin produced with the aid of human manipulation, including, without limitation, Clostridial toxins produced by genetic engineering using random mutagenesis or rational design and Clostridial toxins produced by chemical synthesis. Non-limiting examples of non-naturally occurring Clostridial toxin variants include, e.g., conservative Clostridial toxin variants, non-conservative Clostridial toxin variants, Clostridial toxin chimeric variants and active Clostridial toxin fragments.

As used herein, the term “conservative Clostridial toxin variant” means a Clostridial toxin that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference Clostridial toxin sequence (Table 1). Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative Clostridial toxin variant can function in substantially the same manner as the reference Clostridial toxin on which the conservative Clostridial toxin variant is based, and can be substituted for the reference Clostridial toxin in any aspect of the present invention. A conservative Clostridial toxin variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, 400 or more amino acids, or 500 or more amino acids from the reference Clostridial toxin on which the conservative Clostridial toxin variant is based. A conservative Clostridial toxin variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial toxin on which the conservative Clostridial toxin variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial toxin on which the conservative Clostridial toxin variant is based. Non-limiting examples of a conservative Clostridial toxin variant include, e.g., conservative BoNT/A variants, conservative BoNT/B variants, conservative BoNT/C1 variants, conservative BoNT/D variants, conservative BoNT/E variants, conservative BoNT/F variants, conservative BoNT/G variants, conservative TeNT variants, conservative BaNT variants and conservative BuNT variants.

As used herein, the term “non-conservative Clostridial toxin variant” means a Clostridial toxin in which 1) at least one amino acid is deleted from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based; 2) at least one amino acid added to the reference Clostridial toxin on which the non-conservative Clostridial toxin is based; or 3) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to t h at of the original amino acid from the reference Clostridial toxin sequence (Table 1). A non-conservative Clostridial toxin variant can function in substantially the same manner as the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based, and can be substituted for the reference Clostridial toxin in any aspect of the present invention. A non-conservative Clostridial toxin variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, 400 or more amino acids, or 500 or more amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. Non-limiting examples of a non-conservative Clostridial toxin variant include, e.g., non-conservative BoNT/A variants, non-conservative BoNT/B variants, non-conservative BoNT/C1 variants, non-conservative BoNT/D variants, non-conservative BoNT/E variants, non-conservative BoNT/F variants, non-conservative BoNT/G variants, non-conservative TeNT variants, non-conservative BaNT variants and non-conservative BuNT variants.

As used herein, the term “Clostridial toxin chimeric variant” means a molecule comprising at least a portion of a Clostridial toxin and at least a portion of at least one other protein to form a toxin with at least one property different from the reference Clostridial toxins of Table 1. One class of Clostridial toxin chimeric variant comprises a modified Clostridial toxin were the endogenous cell binding domain of a naturally-occurring Clostridial toxin is either modified or replaced with a cell binding domain of another molecule. Such modified Clostridial toxin possesses an altered cell binding activity because the modified toxin can, e.g., use the same receptor present on the surface of a naturally occurring Clostridial toxin target cell, referred to as an enhanced cell binding activity for a naturally-occurring Clostridial toxin target cell; use a different receptor present on the surface of a naturally occurring Clostridial toxin target cell, referred to as an altered cell binding activity for a naturally-occurring Clostridial toxin target cell, or use a different receptor present on the surface of the non-Clostridial toxin target cell, referred to as an altered cell binding activity for a non-naturally-occurring Clostridial toxin target cell.

A Clostridial toxin chimeric variant can be a modified Clostridial toxin with an enhanced cell binding activity capable of intoxicating a naturally occurring Clostridial toxin target cell, e.g., a motor neuron. One way this enhanced binding activity is achieved by modifying the endogenous targeting domain of a naturally-occurring Clostridial toxin in order to enhance a cell binding activity of the toxin for its naturally-occurring receptor. Such modifications to a targeting domain result in, e.g., a enhanced cell binding activity that increases binding affinity for an endogenous Clostridial toxin receptor present on a naturally-occurring Clostridial toxin target cell; an enhanced cell binding activity that increases binding specificity for a subgroup of endogenous Clostridial toxin receptors present on a naturally-occurring Clostridial toxin target cell; or an enhanced cell binding activity that increases both binding affinity and binding specificity. Non-limiting examples of modified Clostridial toxins an enhanced cell binding activity for a naturally-occurring Clostridial toxin receptor are described in, e.g., Lance E. Steward, et al., Modified Clostridial Toxins with Enhanced Targeting Capabilities For Endogenous Clostridial Toxin Receptors, International Patent Publication No. 2006/008956 (Mar. 14, 2006), Lance E. Steward, Modified Clostridial Toxins with Enhanced Translocation Capability, and Enhanced Targeting Activity, U.S. Provisional Patent Application No. 60/807,063 (Jul. 11, 2006); the content of which are all hereby incorporated by reference in their entirety.

A Clostridial toxin chimeric variant can be a modified Clostridial toxin with an altered cell binding activity capable of intoxicating a naturally occurring Clostridial toxin target cell, e.g., a motor neuron. One way this altered capability is achieved by replacing the endogenous targeting domain of a naturally-occurring Clostridial toxin with a targeting domain of another molecule that selectively binds to a different receptor present on the surface of a naturally occurring Clostridial toxin target cell. Such a modification to a targeting domain results in a modified toxin that is able to selectively bind to a non-Clostridial toxin receptor (target receptor) present on a Clostridial toxin target cell. This enhanced binding activity for a naturally occurring Clostridial toxin target cell allows for lower effective doses of a modified Clostridial toxin to be administered to an individual because more toxin will be delivered to the target cell. Thus, modified Clostridial toxins with an enhanced binding activity will reduce the undesirable dispersal of the toxin to areas not targeted for treatment, thereby reducing or preventing the undesirable side-effects associated with diffusion of a Clostridial toxin to an unwanted location. Non-limiting examples of modified Clostridial toxins with an altered cell binding capability for a Clostridial toxin target cell are described in, e.g., Lance E. Steward et al., Modified Clostridial Toxins with Altered Targeting Capabilities For Clostridial Toxin Target Cells, International Patent Publication No. 2006/009831 (Mar. 14, 2005); Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. patent application Ser. No. 11/376,696 (Mar. 15, 2006); and Lance E. Steward, Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity for Clostridial Toxin Target Cells, U.S. Provisional Patent Application No. 60/807,062, (Jul. 11, 2006); the contents of all of which are hereby incorporated by reference in their entirety.

A Clostridial toxin chimeric variant can be a modified Clostridial toxin with an altered cell binding activity capable of intoxicating a cell other than a naturally occurring Clostridial toxin target cell, e.g., a cell other than a motor neuron. These modified toxins achieve this intoxication by using a target receptor present on non-Clostridial toxin target cell. This re-targeted capability is achieved by replacing a naturally-occurring targeting domain of a Clostridial toxin with a targeting domain showing a selective binding activity for a non-Clostridial toxin receptor present in a non-Clostridial toxin target cell. Such modifications to a targeting domain result in a modified toxin that is able to selectively bind to a non-Clostridial toxin receptor (target receptor) present on a non-Clostridial toxin target cell (re-targeted). A modified Clostridial toxin with an altered targeting activity for a non-Clostridial toxin target cell can bind to a target receptor, translocate into the cytoplasm, and exert its proteolytic effect on the SNARE complex of the non-Clostridial toxin target cell. Non-limiting examples of modified Clostridial toxins with an altered targeting activity for a non-Clostridial toxin target cell are described in, e.g., Keith A. Foster et al., Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent Functions, U.S. Pat. No. 5,989,545 (Nov. 23, 1999); Clifford C. Shone et al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617 (Oct. 8, 2002); Conrad P. Quinn et al., Methods and Compounds for the Treatment of Mucus Hypersecretion, U.S. Pat. No. 6,632,440 (Oct. 14, 2003); Lance E. Steward et al., Methods And Compositions For The Treatment Of Pancreatitis, U.S. Pat. No. 6,843,998 (Jan. 18, 2005); Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Patent Publication 2002/0037833 (Mar. 28, 2002); Keith A. Foster et al., Inhibition of Secretion from Non-neural Cells, U.S. Patent Publication 2003/0180289 (Sep. 25, 2003); J. Oliver Dolly et al., Activatable Recombinant Neurotoxins, International Patent Publication WO 2001/014570 (Mar. 1, 2001); Keith A. Foster et al., Re-targeted Toxin Conjugates, International Patent Publication WO 2005/023309 (Mar. 17, 2005); Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. patent application Ser. No. 11/376,696 (Mar. 15, 2006); Keith A. Foster, Fusion Proteins, International Patent Publication WO 2006/059093 (Jun. 8, 2005); Keith A. Foster, Non-Cytotoxic Protein Conjugates, International Patent Publication WO 2006/059105 (Jun. 8, 2005); and Lance E. Steward, Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Capabilities for Non-Clostridial Toxin Target Cells, U.S. Provisional Patent Application No. 60/807,059, (Jul. 11, 2006); the contents of all of which are hereby incorporated by reference in their entirety. The ability to re-target the therapeutic effects associated with Clostridial toxins has greatly extended the number of medicinal applications able to use a Clostridial toxin therapy. As a non-limiting example, modified Clostridial toxins retargeted to sensory neurons are useful in treating various kinds of chronic pain, such as, e.g., hyperalgesia and allodynia, neuropathic pain and inflammatory pain, see, e.g., Foster, supra, (1999); and Donovan, supra, (2002); and Stephan Donovan, Method For Treating Neurogenic Inflammation Pain with Botulinum Toxin and Substance P Components, U.S. Pat. No. 7,022,329 (Apr. 4, 2006). As another non-limiting example, modified Clostridial toxins retargeted to pancreatic cells are useful in treating pancreatitis, see, e.g., Steward, supra, (2005).

Thus, in an embodiment, a Clostridial toxin chimeric variant can comprise a modified Clostridial toxin disclosed in the present specification where the binding domain comprises an enhanced cell binding activity capable of intoxicating a naturally occurring Clostridial toxin target cell. In another embodiment, a Clostridial toxin chimeric variant can comprise a modified Clostridial toxin disclosed in the present specification where the binding domain comprises an altered cell binding activity capable of intoxicating a naturally occurring Clostridial toxin target cell. In still another embodiment, a Clostridial toxin chimeric variant can comprise a modified Clostridial toxin disclosed in the present specification where the binding domain comprises an altered cell binding activity capable of intoxicating a non-naturally occurring Clostridial toxin target cell.

It is also envisioned that any of a variety of Clostridial toxin fragments can be useful in aspects of the present invention with the proviso that these active fragments can execute the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate. Thus, aspects of this embodiment can include Clostridial toxin fragments having a length of, e.g., at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, at least 1000 amino acids, at least 1100 amino acids and at least 1200 amino acids. Other aspects of this embodiment, can include Clostridial toxin fragments having a length of, e.g., at most 300 amino acids, at most 400 amino acids, at most 500 amino acids, at most 600 amino acids, at most 700 amino acids, at most 800 amino acids, at most 900 amino acids, at most 1000 amino acids, at most 1100 amino acids and at most 1200 amino acids.

It is also envisioned that any of a variety of Clostridial toxin fragments comprising the light chain can be useful in aspects of the present invention with the proviso that these light chain fragments can specifically target the core components of the neurotransmitter release apparatus and thus participate in executing the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate. The light chains of Clostridial toxins are approximately 420-460 amino acids in length and comprise an enzymatic domain (Table 1). Research has shown that the entire length of a Clostridial toxin light chain is not necessary for the enzymatic activity of the enzymatic domain. As a non-limiting example, the first eight amino acids of the BoNT/A light chain (residues 1-8 of SEQ ID NO: 1) are not required for enzymatic activity. As another non-limiting example, the first eight amino acids of the TeNT light chain (residues 1-8 of SEQ ID NO: 8) are not required for enzymatic activity. Likewise, the carboxyl-terminus of the light chain is not necessary for activity. As a non-limiting example, the last 32 amino acids of the BoNT/A light chain (residues 417-448 of SEQ ID NO: 1) are not required for enzymatic activity. As another non-limiting example, the last 31 amino acids of the TeNT light chain (residues 427-457 of SEQ ID NO: 8) are not required for enzymatic activity. Thus, aspects of this embodiment can include Clostridial toxin light chains comprising an enzymatic domain having a length of, e.g., at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids and at least 450 amino acids. Other aspects of this embodiment can include Clostridial toxin light chains comprising an enzymatic domain having a length of, e.g., at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino acids and at most 450 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(N) regions comprising a translocation domain can be useful in aspects of the present invention with the proviso that these active fragments can facilitate the release of the LC from intracellular vesicles into the cytoplasm of the target cell and thus participate in executing the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate. The H_(N) regions from the heavy chains of Clostridial toxins are approximately 410-430 amino acids in length and comprise a translocation domain (Table 1). Research has shown that the entire length of a H_(N) region from a Clostridial toxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include Clostridial toxin H_(N) regions comprising a translocation domain having a length of, e.g., at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include Clostridial toxin H_(N) regions comprising translocation domain having a length of, e.g., at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(C) regions comprising a binding domain can be useful in aspects of the present invention with the proviso that these active fragments can determine the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell execute the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate. The H_(C) regions from the heavy chains of Clostridial toxins are approximately 400-440 amino acids in length and comprise a binding domain (Table 1). Research has shown that the entire length of a H_(C) region from a Clostridial toxin heavy chain is not necessary for the binding activity of the binding domain. Thus, aspects of this embodiment can include Clostridial toxin H_(C) regions comprising a binding domain having a length of, e.g., at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include Clostridial toxin H_(C) regions comprising a binding domain having a length of, e.g., at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.

Thus, in an embodiment, a Clostridial toxin comprises a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain. In an aspect of this embodiment, a Clostridial toxin comprises a naturally occurring Clostridial toxin variant, such as, e.g., a Clostridial toxin isoform or a Clostridial toxin subtype. In another aspect of this embodiment, a Clostridial toxin comprises a non-naturally occurring Clostridial toxin variant, such as, e.g., a conservative Clostridial toxin variant, a non-conservative Clostridial toxin variant or an active Clostridial toxin fragment, or any combination thereof. In another aspect of this embodiment, a Clostridial toxin comprises a Clostridial toxin enzymatic domain or an active fragment thereof, a Clostridial toxin translocation domain or an active fragment thereof, a Clostridial toxin binding domain or an active fragment thereof, or any combination thereof. In other aspects of this embodiment, a Clostridial toxin can comprise a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT or a BuNT.

In another embodiment, a Clostridial toxin comprises a BoNT/A. In an aspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domain, a BoNT/A translocation domain and a BoNT/A binding domain. In another aspect of this embodiment, a BoNT/A comprises SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/A comprises a naturally occurring BoNT/A variant, such as, e.g., a BoNT/A isoform or a BoNT/A subtype. In another aspect of this embodiment, a BoNT/A comprises a naturally occurring BoNT/A variant of SEQ ID NO: 1, such as, e.g., a BoNT/A isoform of SEQ ID NO: 1 or a BoNT/A subtype of SEQ ID NO: 1. In still another aspect of this embodiment, a BoNT/A comprises a non-naturally occurring BoNT/A variant, such as, e.g., a conservative BoNT/A variant, a non-conservative BoNT/A variant or an active BoNT/A fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/A comprises a non-naturally occurring BoNT/A variant of SEQ ID NO: 1, such as, e.g., a conservative BoNT/A variant of SEQ ID NO: 1, a non-conservative BoNT/A variant of SEQ ID NO: 1 or an active BoNT/A fragment of SEQ ID NO: 1, or any combination thereof. In yet another aspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domain or an active fragment thereof, a BoNT/A translocation domain or an active fragment thereof, a BoNT/A binding domain or an active fragment thereof, or any combination thereof. In yet another aspect of this embodiment, a BoNT/A comprising a BoNT/A enzymatic domain of amino acids 1-448 from SEQ ID NO: 1 or an active fragment thereof, a BoNT/A translocation domain of amino acids 449-871 from SEQ ID NO: 1 or an active fragment thereof, a BoNT/A binding domain of amino acids 872-1296 from SEQ ID NO: 1 or an active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 1, at least 75% amino acid identity with the SEQ ID NO: 1, at least 80% amino acid identity with SEQ ID NO: 1, at least 85% amino acid identity with SEQ ID NO: 1, at least 90% amino acid identity with SEQ ID NO: 1 or at least 95% amino acid identity with SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 1, at most 75% amino acid identity with the SEQ ID NO: 1, at most 80% amino acid identity with SEQ ID NO: 1, at most 85% amino acid identity with SEQ ID NO: 1, at most 90% amino acid identity with SEQ ID NO: 1 or at most 95% amino acid identity with SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 1.

In another embodiment, a Clostridial toxin comprises a BoNT/B. In an aspect of this embodiment, a BoNT/B comprises a BoNT/B enzymatic domain, a BoNT/B translocation domain and a BoNT/B binding domain. In another aspect of this embodiment, a BoNT/B comprises SEQ ID NO: 2. In another aspect of this embodiment, a BoNT/B comprises a naturally occurring BoNT/B variant, such as, e.g., a BoNT/B isoform or a BoNT/B subtype. In another aspect of this embodiment, a BoNT/B comprises a naturally occurring BoNT/B variant of SEQ ID NO: 2, such as, e.g., a BoNT/B isoform of SEQ ID NO: 2 or a BoNT/B subtype of SEQ ID NO: 2. In still another aspect of this embodiment, a BoNT/B comprises a non-naturally occurring BoNT/B variant, such as, e.g., a conservative BoNT/B variant, a non-conservative BoNT/B variant or an active BoNT/B fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/B comprises a non-naturally occurring BoNT/B variant of SEQ ID NO: 2, such as, e.g., a conservative BoNT/B variant of SEQ ID NO: 2, a non-conservative BoNT/B variant of SEQ ID NO: 2 or an active BoNT/B fragment of SEQ ID NO: 2, or any combination thereof. In yet another aspect of this embodiment, a BoNT/B comprising a BoNT/B enzymatic domain or an active fragment thereof, a BoNT/B translocation domain or active fragment thereof, a BoNT/B binding domain or active fragment thereof, and any combination thereof. In yet another aspect of this embodiment, a BoNT/B comprising a BoNT/B enzymatic domain of amino acids 1-441 from SEQ ID NO: 2 or active fragment thereof, a BoNT/B translocation domain of amino acids 442-858 from SEQ ID NO: 2 or active fragment thereof, a BoNT/B binding domain of amino acids 859-1291 from SEQ ID NO: 2 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 2, at least 75% amino acid identity with the SEQ ID NO: 2, at least 80% amino acid identity with SEQ ID NO: 2, at least 85% amino acid identity with SEQ ID NO: 2, at least 90% amino acid identity with SEQ ID NO: 2 or at least 95% amino acid identity with SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 2, at most 75% amino acid identity with the SEQ ID NO: 2, at most 80% amino acid identity with SEQ ID NO: 2, at most 85% amino acid identity with SEQ ID NO: 2, at most 90% amino acid identity with SEQ ID NO: 2 or at most 95% amino acid identity with SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 2.

In another embodiment, a Clostridial toxin comprises a BoNT/C1. In an aspect of this embodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain and a BoNT/C1 binding domain. In another aspect of this embodiment, a BoNT/C1 comprises SEQ ID NO: 3. In another aspect of this embodiment, a BoNT/C1 comprises a naturally occurring BoNT/C1 variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1 subtype. In another aspect of this embodiment, a BoNT/C1 comprises a naturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g., a BoNT/C1 isoform of SEQ ID NO: 3 or a BoNT/C1 subtype of SEQ ID NO: 3. In still another aspect of this embodiment, a BoNT/C1 comprises a non-naturally occurring BoNT/C1 variant, such as, e.g., a conservative BoNT/C1 variant, a non-conservative BoNT/C1 variant or an active BoNT/C1 fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/C1 comprises a non-naturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g., a conservative BoNT/C1 variant of SEQ ID NO: 3, a non-conservative BoNT/C1 variant of SEQ ID NO: 3 or an active BoNT/C1 fragment of SEQ ID NO: 3, or any combination thereof. In yet another aspect of this embodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain or active fragment thereof, a BoNT/C1 translocation domain or active fragment thereof, a BoNT/C1 binding domain or active fragment thereof, and any combination thereof. In yet another aspect of this embodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain of amino acid 1-449 from SEQ ID NO: 3 or active fragment thereof, a BoNT/C1 translocation domain of amino acids 450-866 from SEQ ID NO: 3 or active fragment thereof, a BoNT/C1 binding domain of amino acids 867-1291 from SEQ ID NO: 3 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 3, at least 75% amino acid identity with the SEQ ID NO: 3, at least 80% amino acid identity with SEQ ID NO: 3, at least 85% amino acid identity with SEQ ID NO: 3, at least 90% amino acid identity with SEQ ID NO: 3 or at least 95% amino acid identity with SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 3, at most 75% amino acid identity with the SEQ ID NO: 3, at most 80% amino acid identity with SEQ ID NO: 3, at most 85% amino acid identity with SEQ ID NO: 3, at most 90% amino acid identity with SEQ ID NO: 3 or at most 95% amino acid identity with SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 3.

In another embodiment, a Clostridial toxin comprises a BoNT/D. In an aspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain, a BoNT/D translocation domain and a BoNT/D binding domain. In another aspect of this embodiment, a BoNT/D comprises SEQ ID NO: 4. In another aspect of this embodiment, a BoNT/D comprises a naturally occurring BoNT/D variant, such as, e.g., a BoNT/D isoform or a BoNT/D subtype. In another aspect of this embodiment, a BoNT/D comprises a naturally occurring BoNT/D variant of SEQ ID NO: 4, such as, e.g., a BoNT/D isoform of SEQ ID NO: 4 or a BoNT/D subtype of SEQ ID NO: 4. In still another aspect of this embodiment, a BoNT/D comprises a non-naturally occurring BoNT/D variant, such as, e.g., a conservative BoNT/D variant, a non-conservative BoNT/D variant or an active BoNT/D fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/D comprises a non-naturally occurring BoNT/D variant of SEQ ID NO: 4, such as, e.g., a conservative BoNT/D variant of SEQ ID NO: 4, a non-conservative BoNT/D variant of SEQ ID NO: 4 or an active BoNT/D fragment of SEQ ID NO: 4, or any combination thereof. In yet another aspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain or an active fragment thereof, a BoNT/D translocation domain or an active fragment thereof, a BoNT/D binding domain or an active fragment thereof, or any combination thereof. In yet another aspect of this embodiment, a BoNT/D comprising a BoNT/D enzymatic domain of amino acids 1-445 from SEQ ID NO: 4 or an active fragment thereof, a BoNT/D translocation domain of amino acids 446-862 from SEQ ID NO: 4 or an active fragment thereof, a BoNT/D binding domain of amino acids 863-1276 from SEQ ID NO: 4 or an active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 4, at least 75% amino acid identity with the SEQ ID NO: 4, at least 80% amino acid identity with SEQ ID NO: 4, at least 85% amino acid identity with SEQ ID NO: 4, at least 90% amino acid identity with SEQ ID NO: 4 or at least 95% amino acid identity with SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 4, at most 75% amino acid identity with the SEQ ID NO: 4, at most 80% amino acid identity with SEQ ID NO: 4, at most 85% amino acid identity with SEQ ID NO: 4, at most 90% amino acid identity with SEQ ID NO: 4 or at most 95% amino acid identity with SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 4.

In another embodiment, a Clostridial toxin comprises a BoNT/E. In an aspect of this embodiment, a BoNT/E comprises a BoNT/E enzymatic domain, a BoNT/E translocation domain and a BoNT/E binding domain. In another aspect of this embodiment, a BoNT/E comprises SEQ ID NO: 5. In another aspect of this embodiment, a BoNT/E comprises a naturally occurring BoNT/E variant, such as, e.g., a BoNT/E isoform or a BoNT/E subtype. In another aspect of this embodiment, a BoNT/E comprises a naturally occurring BoNT/E variant of SEQ ID NO: 5, such as, e.g., a BoNT/E isoform of SEQ ID NO: 5 or a BoNT/E subtype of SEQ ID NO: 5. In still another aspect of this embodiment, a BoNT/E comprises a non-naturally occurring BoNT/E variant, such as, e.g., a conservative BoNT/E variant, a non-conservative BoNT/E variant or an active BoNT/E fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/E comprises a non-naturally occurring BoNT/E variant of SEQ ID NO: 5, such as, e.g., a conservative BoNT/E variant of SEQ ID NO: 5, a non-conservative BoNT/E variant of SEQ ID NO: 5 or an active BoNT/E fragment of SEQ ID NO: 5, or any combination thereof. In yet another aspect of this embodiment, a BoNT/E comprising a BoNT/E enzymatic domain or an active fragment thereof, a BoNT/E translocation domain or active fragment thereof, a BoNT/E binding domain or active fragment thereof, and any combination thereof. In yet another aspect of this embodiment, a BoNT/E comprising a BoNT/E enzymatic domain of amino acids 1-422 from SEQ ID NO: 5 or active fragment thereof, a BoNT/E translocation domain of amino acids 423-845 from SEQ ID NO: 5 or active fragment thereof, a BoNT/E binding domain of amino acids 846-1252 from SEQ ID NO: 5 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 5, at least 75% amino acid identity with the SEQ ID NO: 5, at least 80% amino acid identity with SEQ ID NO: 5, at least 85% amino acid identity with SEQ ID NO: 5, at least 90% amino acid identity with SEQ ID NO: 5 or at least 95% amino acid identity with SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 5, at most 75% amino acid identity with the SEQ ID NO: 5, at most 80% amino acid identity with SEQ ID NO: 5, at most 85% amino acid identity with SEQ ID NO: 5, at most 90% amino acid identity with SEQ ID NO: 5 or at most 95% amino acid identity with SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 5.

In another embodiment, a Clostridial toxin comprises a BoNT/F. In an aspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain, a BoNT/F translocation domain and a BoNT/F binding domain. In another aspect of this embodiment, a BoNT/F comprises SEQ ID NO: 6. In another aspect of this embodiment, a BoNT/F comprises a naturally occurring BoNT/F variant, such as, e.g., a BoNT/F isoform or a BoNT/F subtype. In another aspect of this embodiment, a BoNT/F comprises a naturally occurring BoNT/F variant of SEQ ID NO: 6, such as, e.g., a BoNT/F isoform of SEQ ID NO: 6 or a BoNT/F subtype of SEQ ID NO: 6. In still another aspect of this embodiment, a BoNT/F comprises a non-naturally occurring BoNT/F variant, such as, e.g., a conservative BoNT/F variant, a non-conservative BoNT/F variant or an active BoNT/F fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/F comprises a non-naturally occurring BoNT/F variant of SEQ ID NO: 6, such as, e.g., a conservative BoNT/F variant of SEQ ID NO: 6, a non-conservative BoNT/F variant of SEQ ID NO: 6 or an active BoNT/F fragment of SEQ ID NO: 6, or any combination thereof. In yet another aspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain or active fragment thereof, a BoNT/F translocation domain or active fragment thereof, a BoNT/F binding domain or active fragment thereof, and any combination thereof. In yet another aspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain of amino acid 1-439 from SEQ ID NO: 6 or active fragment thereof, a BoNT/F translocation domain of amino acids 440-864 from SEQ ID NO: 6 or active fragment thereof, a BoNT/F binding domain of amino acids 865-1274 from SEQ ID NO: 6 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 6, at least 75% amino acid identity with the SEQ ID NO: 6, at least 80% amino acid identity with SEQ ID NO: 6, at least 85% amino acid identity with SEQ ID NO: 6, at least 90% amino acid identity with SEQ ID NO: 6 or at least 95% amino acid identity with SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 6, at most 75% amino acid identity with the SEQ ID NO: 6, at most 80% amino acid identity with SEQ ID NO: 6, at most 85% amino acid identity with SEQ ID NO: 6, at most 90% amino acid identity with SEQ ID NO: 6 or at most 95% amino acid identity with SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 6.

In another embodiment, a Clostridial toxin comprises a BoNT/G. In an aspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domain, a BoNT/G translocation domain and a BoNT/G binding domain. In another aspect of this embodiment, a BoNT/G comprises SEQ ID NO: 7. In another aspect of this embodiment, a BoNT/G comprises a naturally occurring BoNT/G variant, such as, e.g., a BoNT/G isoform or a BoNT/G subtype. In another aspect of this embodiment, a BoNT/G comprises a naturally occurring BoNT/G variant of SEQ ID NO: 7, such as, e.g., a BoNT/G isoform of SEQ ID NO: 7 or a BoNT/G subtype of SEQ ID NO: 7. In still another aspect of this embodiment, a BoNT/G comprises a non-naturally occurring BoNT/G variant, such as, e.g., a conservative BoNT/G variant, a non-conservative BoNT/G variant or an active BoNT/G fragment, or any combination thereof. In still another aspect of this embodiment, a BoNT/D comprises a non-naturally occurring BoNT/G variant of SEQ ID NO: 7, such as, e.g., a conservative BoNT/G variant of SEQ ID NO: 7, a non-conservative BoNT/G variant of SEQ ID NO: 7 or an active BoNT/G fragment of SEQ ID NO: 7, or any combination thereof. In yet another aspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domain or an active fragment thereof, a BoNT/G translocation domain or an active fragment thereof, a BoNT/G binding domain or an active fragment thereof, or any combination thereof. In yet another aspect of this embodiment, a BoNT/G comprising a BoNT/G enzymatic domain of amino acids 1-446 from SEQ ID NO: 7 or an active fragment thereof, a BoNT/G translocation domain of amino acids 447-863 from SEQ ID NO: 7 or an active fragment thereof, a BoNT/G binding domain of amino acids 864-1297 from SEQ ID NO: 7 or an active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 7, at least 75% amino acid identity with the SEQ ID NO: 7, at least 80% amino acid identity with SEQ ID NO: 7, at least 85% amino acid identity with SEQ ID NO: 7, at least 90% amino acid identity with SEQ ID NO: 7 or at least 95% amino acid identity with SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 7, at most 75% amino acid identity with the SEQ ID NO: 7, at most 80% amino acid identity with SEQ ID NO: 7, at most 85% amino acid identity with SEQ ID NO: 7, at most 90% amino acid identity with SEQ ID NO: 7 or at most 95% amino acid identity with SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 7.

In another embodiment, a Clostridial toxin comprises a TeNT. In an aspect of this embodiment, a TeNT comprises a TeNT enzymatic domain, a TeNT translocation domain and a TeNT binding domain. In an aspect of this embodiment, a TeNT comprises SEQ ID NO: 8. In another aspect of this embodiment, a TeNT comprises a naturally occurring TeNT variant, such as, e.g., a TeNT isoform or a TeNT subtype. In another aspect of this embodiment, a TeNT comprises a naturally occurring TeNT variant of SEQ ID NO: 8, such as, e.g., a TeNT isoform of SEQ ID NO: 8 or a TeNT subtype of SEQ ID NO: 8. In still another aspect of this embodiment, a TeNT comprises a non-naturally occurring TeNT variant, such as, e.g., a conservative TeNT variant, a non-conservative TeNT variant or an active TeNT fragment, or any combination thereof. In still another aspect of this embodiment, a TeNT comprises a non-naturally occurring TeNT variant of SEQ ID NO: 8, such as, e.g., a conservative TeNT variant of SEQ ID NO: 8, a non-conservative TeNT variant of SEQ ID NO: 8 or an active TeNT fragment of SEQ ID NO: 8, or any combination thereof. In yet another aspect of this embodiment, a TeNT comprising a TeNT enzymatic domain or an active fragment thereof, a TeNT translocation domain or active fragment thereof, a TeNT binding domain or active fragment thereof, and any combination thereof. In yet another aspect of this embodiment, a TeNT comprising a TeNT enzymatic domain of amino acids 1-457 from SEQ ID NO: 8 or active fragment thereof, a TeNT translocation domain of amino acids 458-879 from SEQ ID NO: 8 or active fragment thereof, a TeNT binding domain of amino acids 880-1315 from SEQ ID NO: 8 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 8, at least 75% amino acid identity with the SEQ ID NO: 8, at least 80% amino acid identity with SEQ ID NO: 8, at least 85% amino acid identity with SEQ ID NO: 8, at least 90% amino acid identity with SEQ ID NO: 8 or at least 95% amino acid identity with SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 8, at most 75% amino acid identity with the SEQ ID NO: 8, at most 80% amino acid identity with SEQ ID NO: 8, at most 85% amino acid identity with SEQ ID NO: 8, at most 90% amino acid identity with SEQ ID NO: 8 or at most 95% amino acid identity with SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 8. In other aspects of this embodiment, a TeNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 8.

In another embodiment, a Clostridial toxin comprises a BaNT. In an aspect of this embodiment, a BaNT comprises a BaNT enzymatic domain, a BaNT translocation domain and a BaNT binding domain. In another aspect of this embodiment, a BaNT comprises SEQ ID NO: 9. In another aspect of this embodiment, a BaNT comprises a naturally occurring BaNT variant, such as, e.g., a BaNT isoform or a BaNT subtype. In another aspect of this embodiment, a BaNT comprises a naturally occurring BaNT variant of SEQ ID NO: 9, such as, e.g., a BaNT isoform of SEQ ID NO: 9 or a BaNT subtype of SEQ ID NO: 9. In still another aspect of this embodiment, a BaNT comprises a non-naturally occurring BaNT variant, such as, e.g., a conservative BaNT variant, a non-conservative BaNT variant or an active BaNT fragment, or any combination thereof. In still another aspect of this embodiment, a BaNT comprises a non-naturally occurring BaNT variant of SEQ ID NO: 9, such as, e.g., a conservative BaNT variant of SEQ ID NO: 9, a non-conservative BaNT variant of SEQ ID NO: 9 or an active BaNT fragment of SEQ ID NO: 9, or any combination thereof. In yet another aspect of this embodiment, a BaNT comprises a BaNT enzymatic domain or an active fragment thereof, a BaNT translocation domain or an active fragment thereof, a BaNT binding domain or an active fragment thereof, or any combination thereof. In yet another aspect of this embodiment, a BaNT comprising a BaNT enzymatic domain of amino acids 1-448 from SEQ ID NO: 9 or an active fragment thereof, a BaNT translocation domain of amino acids 449-871 from SEQ ID NO: 9 or an active fragment thereof, a BaNT binding domain of amino acids 872-1296 from SEQ ID NO: 9 or an active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 9, at least 75% amino acid identity with the SEQ ID NO: 9, at least 80% amino acid identity with SEQ ID NO: 9, at least 85% amino acid identity with SEQ ID NO: 9, at least 90% amino acid identity with SEQ ID NO: 9 or at least 95% amino acid identity with SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 9, at most 75% amino acid identity with the SEQ ID NO: 9, at most 80% amino acid identity with SEQ ID NO: 9, at most 85% amino acid identity with SEQ ID NO: 9, at most 90% amino acid identity with SEQ ID NO: 9 or at most 95% amino acid identity with SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 9. In other aspects of this embodiment, a BaNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 9.

In another embodiment, a Clostridial toxin comprises a BuNT. In an aspect of this embodiment, a BuNT comprises a BuNT enzymatic domain, a BuNT translocation domain and a BuNT binding domain. In another aspect of this embodiment, a BuNT comprises SEQ ID NO: 10. In another aspect of this embodiment, a BuNT comprises a naturally occurring BuNT variant, such as, e.g., a BuNT isoform or a BuNT subtype. In another aspect of this embodiment, a BuNT comprises a naturally occurring BuNT variant of SEQ ID NO: 10, such as, e.g., a BuNT isoform of SEQ ID NO: 10 or a BuNT subtype of SEQ ID NO: 10. In still another aspect of this embodiment, a BuNT comprises a non-naturally occurring BuNT variant, such as, e.g., a conservative BuNT variant, a non-conservative BuNT variant or an active BuNT fragment, or any combination thereof. In still another aspect of this embodiment, a BuNT comprises a non-naturally occurring BuNT variant of SEQ ID NO: 10, such as, e.g., a conservative BuNT variant of SEQ ID NO: 10, a non-conservative BuNT variant of SEQ ID NO: 10 or an active BuNT fragment of SEQ ID NO: 10, or any combination thereof. In yet another aspect of this embodiment, a BuNT comprises a BuNT enzymatic domain or an active fragment thereof, a BuNT translocation domain or an active fragment thereof, a BuNT binding domain or an active fragment thereof, or any combination thereof. In yet another aspect of this embodiment, a BuNT comprising a BuNT enzymatic domain of amino acids 1-448 from SEQ ID NO: 10 or an active fragment thereof, a BuNT translocation domain of amino acids 449-871 from SEQ ID NO: 10 or an active fragment thereof, a BuNT binding domain of amino acids 872-1296 from SEQ ID NO: 10 or an active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 10, at least 75% amino acid identity with the SEQ ID NO: 10, at least 80% amino acid identity with SEQ ID NO: 10, at least 85% amino acid identity with SEQ ID NO: 10, at least 90% amino acid identity with SEQ ID NO: 10 or at least 95% amino acid identity with SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 10, at most 75% amino acid identity with the SEQ ID NO: 10, at most 80% amino acid identity with SEQ ID NO: 10, at most 85% amino acid identity with SEQ ID NO: 10, at most 90% amino acid identity with SEQ ID NO: 10 or at most 95% amino acid identity with SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid substitutions relative to SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletions relative to SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions relative to SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 10. In other aspects of this embodiment, a BuNT comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO: 10.

Using this information it is possible to construct an expressible open nucleic acid reading frame for insertion into an expression vector and subsequent expression within a chosen host cell. Indeed, International Patent publication WO 01/14570 discloses methods of making single-chain, cleavable recombinant modified or unmodified Clostridial neurotoxin derivatives and chimeric and hybrid forms thereof using such methods. Additional publications disclosing methods of making expressible recombinant neurotoxins and derivatives thereof include U.S. Pat. Nos. 5,989,545; 6,203,794; 6,395,513; U.S. Publication Numbers U.S. 2003/0166238; U.S. 2002/169942; U.S. 2004/176299; U.S. 2004/126397; U.S. 2005/035730; U.S. 2005/068494; U.S. 2006/011966; International Patent Applications W095/32738; WO 99/55359; W096/33273; W098/07864; W099/17806; WO98/07864; WO02/44199; WO02/40506. All these publications are incorporated by reference herein in their entirety.

The use of recombinant DNA techniques permits the construction of modified clostridial neurotoxins having different functional properties from the naturally occurring toxin subtypes and strains thereof. For example, altering the naturally occurring amino acid sequence of the native neurotoxin light chain and/or adding a different therapeutic moiety permits the construction of transport proteins designed to carry a therapeutic agent within a neuron. See U.S. Pat. No. 6,203,794 (incorporated by reference herein). Altering the targeting (binding) domain permits the toxin to be transported within pancreatic cells, such as acinar cells, thereby preventing secretion of activated digestive enzymes by such cells, See U.S. Pat. No. 6,843,998 (hereby incorporated by reference herein), or sensory afferent neurons, thereby preventing neurotransmitter release and thus providing relief from pain; see U.S. Pat. No. 6,395,513 (hereby incorporated by reference herein.)

In addition, the creation of chimeric neurotoxin derivatives comprising, for example, the binding domain and the translocation domain (or modified versions thereof) of one neurotoxin subtype for example, BoNT/A, and the light chain region of another neurotoxin subtype, for example, BoNT/E. It will be seen that given the general structural homology between the neurotoxin subtypes, any combination of the three basic clostridial neurotoxin domains, may be made in a single amino acid chain (or cleaved di-chain molecule). Thus, a binding region from any of neurotoxin BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT or BuNT may be independently combined with a translocation domain from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT or BuNT, and further independently combined with a endopeptidase domain from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT or BuNT.

The therapeutic utility of a Clostridial toxin may be enhanced or altered by the inclusion of at least two cell binding domains. All such modified Clostridial toxins are referred to herein as “multivalent Clostridial toxin.” Each binding domain of a multivalent Clostridial toxin may comprise any binding domain capable of binding under physiological conditions to a cell surface receptor capable of selectively binding to the ligand. Furthermore, each binding domain is capable of selectively binding to a receptor present on the surface of a target cell, or facilitating the entry of the multivalent Clostridial toxin into a target cell. In addition, at least one of said binding domains will bind selectively to the target cell and at least one of the binding domains will bind to a cell surface receptor capable of mediating the internalization of the multivalent Clostridial toxin within the target cell. By adding, for example, multiple binding domains the binding constant (Kd) (which equals [toxin][target cell]/[toxin:target cell complex]) of the multivalent Clostridial toxin will be increased over that of an unmodified toxin or monovalent toxin.

Aspects of the present invention provide, in part, a binding domain. As used herein, the term “binding domain” is synonymous with “ligand” and means any molecule that can selectively interact with another molecule present on the surface of a cell and initiate the overall internalization mechanism whereby the multivalent Clostridial toxin disclosed in the present specification intoxicates a target cell. Each of the two or more binding domains in a multivalent Clostridial neurotoxin may be, for example, a binding domain that facilitates stability, solubility and/or cell penetration. At least one such binding domain is selective for a cell surface molecule of a target cell. In addition, at least one cell surface molecule to which a binding domain of a multivalent Clostridial neurotoxin binds is capable of being internalized by the target cell after binding. Preferably, at least one cell surface molecule to which a binding domain of a multivalent Clostridial neurotoxin selectively binds is capable of being internalized by the target cell after binding. As used herein, the term “selectively” means having a highly preferred activity or effect. As used herein, the term “selectively bind” or “selective binding” means a molecule is able to bind its target receptor under physiological conditions, or in vitro conditions substantially approximating physiological conditions, to a statistically significantly greater degree relative to other, non-target receptors. Thus, with reference to a binding domain or ligand of the present specification, there is a discriminatory binding of the binding domain to a cell surface receptor present on a cell. Thus, in an embodiment, a binding domain or ligand can selectively bind a cell surface receptor with a dissociation constant (Kd) of the ligand for target receptor by at least four fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 1000, at least 100,000 fold the value of Kd of the ligand for other cell surface receptors.

A binding domain disclosed in the present specification may facilitate the binding activity of a multivalent Clostridial toxin to a molecule located at the surface of a cell. As used herein, the term “binding activity” means that one molecule is directly or indirectly contacting another molecule via at least one intermolecular or intramolecular force, including, without limitation, a covalent bond, an ionic bond, a metallic bond, a hydrogen bond, a hydrophobic interaction, a van der Waals interaction, and the like, or any combination thereof. “Bound” and “bind” are considered terms for binding.

As used herein, the term “binding affinity” means how strong a binding domain's activity is for a particular cell surface molecule. In general, high binding affinity results from greater intermolecular force between a binding domain and its cell surface molecule while low binding affinity involves less intermolecular force between the ligand and its cell surface molecule. High binding affinity involves a longer residence time for the binding domain at its cell surface molecule binding site than is the case for low binding affinity. As such, a binding domain with a high binding affinity means a lower concentration of that binding domain is required to maximally occupy the binding sites of a cell surface molecule and trigger a physiological response. Conversely, low binding affinity means a relatively high concentration of a binding domain is required before the binding sites of a cell surface molecule is maximally occupied and the maximum physiological response is achieved. Thus, multivalent Clostridial toxins with increased binding activity due to high binding affinity will allow administration of reduced doses of the toxin, thereby reducing or preventing unwanted side-effects associated with toxin dispersal into non-targeted areas.

As used herein, the term “binding specificity” means how specific a binding domain's activity is one particular cell surface molecule. In general, high binding specificity results in a more exclusive interaction with one particular cell surface molecule or subgroup of cell surface molecules while low binding specificity results in a more promiscuous interaction with a larger group of cell surface molecules. As such, a binding domain with a high binding specificity means that binding domain will occupy the binding sites of a particular cell surface molecule and trigger a physiological response. Conversely, low binding specificity means a binding domain will occupy the binding sites of many cell surface molecules and trigger a multitude of physiological responses. Thus, multivalent Clostridial toxins with increased binding activity due to high binding specificity will only target cell surface molecules present on a subgroup of target cells, thereby reducing the side effects associated with the targeting of all target cells.

As used herein, the term “cell surface molecule” means both a traditional receptor capable of binding a ligand and thereby causing a ligand-selective response (at e.g., the cell surface, the cytoplasm, or both), and a cell surface feature such as caveolae, membrane “patches”, or lipid rafts, which appear to serve at least in part to gather together and locally concentrate at the cell surface specific receptors and bound ligand types for later processing such as endocytosis, often mediated by a cell surface receptor.

Thus, in an embodiment, a multivalent Clostridial toxin can comprises a plurality of binding domains. In aspects of this embodiment, a multiivalent Clostridal toxion can comprise, e.g., at least two binding domains, at least three binding domains, at least four binding domains or at least five binding domains. In other aspects of this embodiment, a multiivalent Clostridal toxion can comprise, e.g., at most two binding domains, at most three binding domains, at most four binding domains or at most five binding domains. In other aspects of this embodiment, a multiivalent Clostridal toxion comprises, e.g., two binding domains, three binding domains, four binding domains or five binding domains.

It is envisioned that any and all binding domains capable of binding a Clostridial toxin receptor present on a naturally-occurring Clostridial toxin target cell can be used to practice aspects of the present invention, including, without limitation, a Clostridial toxin binding domain, such as, e.g., a BoNT/A binding domain, a BoNT/B binding domain, a BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E binding domain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT binding domain, a BaNT binding domain, and a BuNT binding domain; a Clostridial non-toxin associated protein, such as, e.g., a BoNT/A HA-33, a BoNT/B HA-33, a BoNT/C1 HA-33, a BoNT/D HA-33, a BoNT/A HA-17, a BoNT/B HA-17, a BoNT/C1 HA-17, a BoNT/D HA-17, a BoNT/A NTNH, a BoNT/B NTNH, a BoNT/C1 NTNH, a BoNT/D NTNH, a BoNT/E NTNH, a BoNT/F NTNH and a BoNT/G NTNH; and a FGF, such as, e.g., a FGF-1, a FGF-2, a FGF-4, a FGF-8, a FGF-9, a FGF-17 and a FGF-18.

It is envisioned that any and all binding domains capable of binding a non-Clostridial toxin receptor present on a naturally-occurring Clostridial toxin target cell can be used to practice aspects of the present invention, including, without limitation, polypeptides that selectively bind to a receptor present on a presynaptic membrane and polypeptides that selectively bind to a receptor present on a postsynaptic membrane. Polypeptides that appear to bind to a receptor present on a presynaptic membrane, include, without limitation, Glucagon like hormones, such as, e.g., secretin, Ghrelin (GHS), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), pituitary adenylate cyclase activating peptide (PACAP), glicentin, glicentin-related polypeptide (GRPP), oxyntomodulin (OXY), vasoactive intestinal peptide-1 (VIP-1), vasoactive intestinal peptide-2 (VIP-2), gastric inhibitory polypeptide (GIP), a galanin (Gal) or a calcitonin-related peptidesvisceral gut peptide; neurohormones, such as, e.g., corticotropin-releasing hormone (CCRH) and parathyroid hormone (PTH); neuroregulatory cytokines, such as, e.g., ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemia inhibitory factor (LIF), an interleukin (IL), onostatin M (OSM), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), neuroleukin (NL), VEGF, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2) and epidermal growth factor (EGF); neurotrophins, such as, e.g., nerve growth factors (NGFs), brain-derived growth factors (BDNFs), neurotrophin-3s (NT-3s) and neurotrophin-4/5s (NT-4/5s); growth factors, such as, e.g., glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), persephrin (PSPN), artemin (ARTN); transformation growth factor betas (TGFβs), bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), activins; axon guidance signaling molecules, such as, e.g., netrins, semaphrorings and ephrins; sugar binding proteins, such as, e.g., serum amyloid P, β-glucanase, sialidase, lectin, cryia, insecticidal delta-endotoxin, agglutinin, abrin and ricin; ligands that selectively bind neurexins, such as, e.g., ligands for neurexin-1α and neurexin-1β; ligands for neurexin-2α and neurexin-2β; and ligands for neurexin-3α and neurexin-3β; and WNTs. Ligands that appear to bind to a receptor present on a postsynaptic membrane, include, without limitation, Ng-CAM (L1), NCAM, N-cadherin, Agrin-MUSK, basement membrane polypeptides, such as, e.g., laminin β-2.

It is envisioned that any and all binding domains capable of binding a non-Clostridial toxin receptor present on a non-Clostridial toxin target cell can be used to practice aspects of the present invention, including, without limitation, polypeptides that selectively bind to a receptor present on a sensory neuron, an autonomic neuron or a non-neuronal cell. Such binding domains include, without limitation, an opioid peptide, such as, e.g., an enkephalin, a bovine adrenomedullary-22 (BAM22) peptide, an endomorphin, an endorphin, a dynorphin, a nociceptin or a hemorphin; a melanocortin peptide, such as, e.g., an α-melanocyte stimulating hormones (α-MSH), a β-melanocyte stimulating hormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH), an adrenocorticotropin (ACTH), a Corticotropin-like intermediary peptide (CLIP), a β-lipotropin (β-LPH) and a γ-lipotropin (γ-LPH); a galanin, such as, e.g., a galanin and a galanin message-associated peptide (GMAP); a granin, such as, e.g., a chromogranin A peptide like a β-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, a catestatin, a parastatin and a GE-25, a chromogranin B (secretogranin I) peptide like a GAWK peptide, an adrenomedullary peptide and a secretolytin and a chromogranin C (secretogranin II) peptide like secretoneurin, EM66 and manserin; a tachykinin peptide, such as, e.g., Substance P, neuropeptide K (NPK), neuropeptide gamma (NP gamma), neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), neurokinin B (NKB), a hemokinin and a endokinin; a cholecystokinin peptide, such as, e.g., a cholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, a cholecystokinin 12 and a cholecystokinin 8; a Neuropeptide Y related peptide, such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) and a Pancreatic icosapeptide (PIP); a kinin peptide, such as, e.g., a bradykinin, a kallidan, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin; a protease activated receptor (PAR) peptide, such as, e.g., a PAR1 peptide, a PAR2 peptide, a PAR3 peptide and a PAR4 peptide; a corticotropin-releasing hormone; a thyrotropin-releasing hormone; a somatostatin; a leukemia inhibitor factor (LIF); and an interleukin-1 (IL1).

It is envisioned that any and all binding domains capable of facilitating the transport of the multivalent Clostridial toxin across a cell membrane of a target cell can be used to practice aspects of the present invention, including, without limitation, a translocator like a protein translocation domain (PTD), such as, e.g., a herpes simplex virus type 1 VP22 protein translocating sequence, a SV-40 virus large T translocating sequence, a TAT translocating sequence, an adenovirus translocating sequence, a synthetic integrin binding domain translocating sequence, a Kaposi fibroblast growth factor membrane translocating sequence, a nuclear localization signal, a Transportan translocating sequence, a ciliary neurotrophic factor translocating sequence, a caveolin, an interleukin 1-β translocating sequence, a thioredoxin translocating sequence, a fibroblast growth factor-1 translocating sequence, a fibroblast growth factor-2 translocating sequence, an integrin β1 translocating sequence, an integrin β3 translocating sequence, a lactoferrin translocating sequence, a homeodomain translocating sequence, like, a penetratin translocating sequence, an Engrailed-1 translocating sequence, an Engrailed-2 translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4 translocating sequence, a Hoxc-8 translocating sequence

It is envisioned that any and all binding domains capable of binding to a cell surface molecule capable of facilitating the transport of a multivalent Clostridial toxin across a cell membrane of a target cell can be used to practice aspects of the present invention, including, without limitation, an antibody to a coated pit protein, such as, e.g., a clatherin antibody and an Adaptor Protein-2 (adaptin) antibody; an antibody to a caveolae-associated protein, such as, e.g., a caveolin-1 antibody and a GPI-linked receptor protein antibody.

An example of a binding domain, includes, without limitation, a Clostridial toxin binding domain such, as, e.g., a BoNT/A binding domain, a BoNT/B binding domain, a BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E binding domain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT binding domain, a BaNT binding domain and a BuNT binding domain

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C) domain of TeNT indicate that the carboxyl-terminal H_(CC) domain comprises a modified β-trefoil domain which forms three distinct carbohydrate binding regions domains that resembles the carbohydrate binding moiety found in many sugar-binding proteins, such as, e.g., serum amyloid P, sialidase, cryia, insecticidal ∂-endotoxin and lectins (FIG. 3). Biochemical studies indicate that the β-trefoil domain structure of the H_(CC) domain appears to mediate the binding to specific carbohydrate containing components of the Clostridial toxin receptor on the cell surface, see, e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family Provides Residue Level Information About the Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124 (2000).

Proteins containing the structural β-trefoil domain represents a diverse group of proteins, see, e.g., C. A. Orengo et al., Protein Superfamilies and Domain Superfolds, 372 Nature 631-634 (1994). The β-trefoil domain comprises a six-stranded β-barrel closed off at one end by three β-hairpin structures that exhibits a characteristic pseudo-threefold axis symmetry. The monomeric structural unit of this three-fold symmetry is referred to as the β-trefoil fold that contains four β-sheets organized as a pair of antiparallel β-sheets. Dividing each of these β-trefoil folds is a β-hairpin turn. Therefore, in a linear fashion, a β-trefoil domain comprises four β-sheets of the first β-trefoil fold (α-fold), a β-hairpin turn, four β-sheets of the second β-trefoil fold (β-fold), a second β-hairpin turn four β-sheets of the third β-trefoil fold (γ-fold) (see FIG. 2). Because the first hairpin turn is located between the fourth and fifth β-sheets of the β-trefoil domain, it is designated the β4/β5 β-hairpin turn. Likewise, since the second hairpin turn is located between the eight and ninth β-sheets of the β-trefoil domain, it is designated the β8/β9 β-hairpin turn.

TABLE 2 β-trefoil Domains of Clostridial Toxins Amino Acid Sequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold BoNT/A 1 1111-1162 1163-1178 1179-1223 1224-1236 1237-1296 BoNT/B 2 1098-1147 1148-1165 1166-1210 1211-1222 1223-1291 BoNT/C1 3 1112-1150 1151-1166 1167-1218 1219-1229 1230-1291 BoNT/D 4 1099-1137 1138-1153 1154-1207 1208-1218 1219-1276 BoNT/E 5 1086-1129 1130-1146 1147-1190 1191-1198 1199-1252 BoNT/F 6 1106-1152 1153-1171 1172-1213 1214-1221 1222-1274 BoNT/G 7 1106-1153 1154-1172 1173-1218 1219-1230 1231-1297 TeNT 8 1128-1177 1178-1194 1195-1240 1241-1254 1255-1315 BaNT 9 1095-1142 1143-1161 1162-1207 1208-1215 1216-1268 BuNT 10  1086-1129 1130-1146 1147-1190 1191-1197 1198-1251

Continuing research has elucidated that β4/β5 and β8/β9 β-hairpin turns are important in conferring the proper pseudo-threefold axis symmetry observed in the β-trefoil domain. Additionally, this work has demonstrated that amino acid changes in these two β-hairpin turns can increase the stability of the β-trefoil domain, which in turn results in increased binding activity, see, e.g., Stephen R. Brych et al., Structure and Stability Effects of Mutations Designed to Increase the Primary Sequence Symmetry Within the Core Region of a β-trefoil, 10 Protein Sci. 2587-2599 (2001); Jaewon Kim et al., Alternative Type I and I′ Turn Conformations in the β8/β9 β-hairpin of Human Acidic Fibroblast Growth Factor, 11 Protein Sci. 459-466 (2002); Jaewon Kim et al., Sequence swapping Does Not Result in Conformation Swapping for the β4/β5 and β8/β9 β-hairpin Turns in Human Acidic Fibroblast Growth Factor, 14 Protein Sci. 351-359 (2005). As a non-limiting example, replacement of an amino acid comprising either the β4/β5 hairpin turn or β8/β9 β-hairpin turn with a glycine results in increased stabilization of the β-trefoil domain. Therefore, replacement of amino acids located in the β4/β5 and β8/β9 β-hairpin turns of the β-trefoil domains present in the binding domain of Clostridial toxins will increase binding activity of such a modified Clostridial toxin by increasing the structural stability of the β-trefoil domain. The amino acid sequences comprising the β-trefoil domains found in various Clostridial toxins are shown in Table 2.

As is typical for proteins containing a β-trefoil fold, the overall amino acid sequence identity of the H_(CC) domain between Clostridial toxins is low. However, key residues essential for binding activity have been identified by structural analysis and mutagenesis experiments, see, e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family Provides Residue Level Information About the Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124 (2000). For example, analysis of the H_(CC) domain structure by crystallography identified five highly conserved residues critical for forming a shallow surface pocket of a carbohydrate binding moiety. These polar residues make hydrogen bonds with the carbohydrate ring. In BoNT/A these five polar residues are Glu 1203, Phe 1252, Ser 1264, Tyr 1267 and Gly 1279, while in TeNT, these residues are Asp 1222, Thr 1270, Ser 1287, Tyr 1290 and Gly 1300. Additionally, tyrosine residues forming the hydrophilic wall of this pocket were also important (Trp 1266 of BoNT/A and Trp 1289 of TeNT) and tryptophan fluorescence quenching experiments indicated that Trp 1266 of BoNT/A bound carbohydrate molecules. In another studies, photoaffinity labeling experiments revealed that Gln 1270 of BoNT/A and His 1293 of TeNT were also involved in binding carbohydrate molecules. Mutagenesis experiments designed to assay loss-of-function binding activity mutations confirmed the importance of many of the residues described above for BoNT/A and TeNT and extended this analysis to BoNT/B (Glu 1190, His 1241, Typ 1262, Tyr 1263), see, e.g., Andreas Rummel et al., The H _(CC)-Domain of Botulinum Neurotoxins A and B Exhibits a Singular Ganglioside Binding Site Displaying Serotype Specific Carbohydrate Interaction, 51(3) Mol. Microbiol. 631-643 (2004).

As used herein, the term “Clostridial toxin binding domain” means any Clostridial toxin polypeptide that can execute the cell binding step of the intoxication process, including, e.g., the selective binding of the Clostridial toxin to a toxin-specific receptor located on the plasma membrane surface of a target cell. Non-limiting examples of a Clostridial toxin binding domain include, e.g., a Clostridial toxin H_(C) binding domain, such as, e.g., a BoNT/A H_(C) binding domain, a BoNT/B H_(C) binding domain, a BoNT/C1 H_(C) binding domain, a BoNT/D H_(C) binding domain, a BoNT/E H_(C) binding domain, a BoNT/F H_(C) binding domain, a BoNT/G H_(C) binding domain, a TeNT H_(C) binding domain, a BaNT H_(C) binding domain and a BuNT H_(C) binding domain. Other non-limiting examples of a Clostridial toxin H_(CC) binding domain, include a BoNT/A H_(CC) binding domain, a BoNT/B H_(CC) binding domain, a modified BoNT/C1 H_(CC) binding domain, a BoNT/D H_(CC) binding domain, a BoNT/E H_(CC) binding domain, a BoNT/F H_(CC) binding domain, a BoNT/G H_(CC) binding domain, a TeNT H_(CC) binding domain, a BaNT H_(CC) binding domain and a BuNT H_(CC) binding domain.

A Clostridial toxin binding domain includes, without limitation, naturally occurring Clostridial toxin binding domain variants, such as, e.g., Clostridial toxin binding domain isoforms and Clostridial toxin binding domain subtypes; non-naturally occurring Clostridial toxin binding domain variants, such as, e.g., conservative Clostridial toxin binding domain variants, non-conservative Clostridial toxin binding domain variants, Clostridial toxin binding domain chimerics, active Clostridial toxin binding domain fragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin binding domain variant,” whether naturally-occurring or non-naturally-occurring, means a Clostridial toxin binding domain that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Table 1) and can be described in percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all Clostridial toxin binding domain variants disclosed in the present specification are capable of executing the cell binding step of the intoxication process, including, e.g., the selective binding of the Clostridial toxin to a toxin-specific receptor located on the plasma membrane surface of a target cell.

As a non-limiting example, a BoNT/A H_(C) binding domain variant comprising amino acids 874-1296 of SEQ ID NO: 1 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 874-1296 of SEQ ID NO: 1; a BoNT/B H_(C) binding domain variant comprising amino acids 861-1291 of SEQ ID NO: 2 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 861-1291 of SEQ ID NO: 2; a BoNT/C1 H_(C) binding domain variant comprising amino acids 869-1291 of SEQ ID NO: 3 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 869-1291 of SEQ ID NO: 3; a BoNT/D H_(C) binding domain variant comprising amino acids 865-1276 of SEQ ID NO: 4 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 865-1276 of SEQ ID NO: 4; a BoNT/E H_(C) binding domain variant comprising amino acids 848-1252 of SEQ ID NO: 5 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 848-1252 of SEQ ID NO: 5; a BoNT/F H_(C) binding domain variant comprising amino acids 867-1274 of SEQ ID NO: 6 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 867-1274 of SEQ ID NO: 6; a BoNT/G H_(C) binding domain variant comprising amino acids 866-1297 of SEQ ID NO: 7 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 866-1297 of SEQ ID NO: 7; a TeNT H_(C) binding domain variant comprising amino acids 882-1315 of SEQ ID NO: 8 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 882-1315 of SEQ ID NO: 8, a BaNT H_(C) binding domain variant comprising amino acids 858-1269 of SEQ ID NO: 9 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 858-1268 of SEQ ID NO: 9, and a BuNT H_(C) binding domain variant comprising amino acids 848-1251 of SEQ ID NO: 10 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 848-1251 of SEQ ID NO: 10.

As another non-limiting example, a BoNT/A H_(CC) binding domain comprising amino acids 1092-1296 of SEQ ID NO: 1 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1092-1296 of SEQ ID NO: 1; a modified BoNT/B H_(CC) binding domain comprising amino acids 1079-1291 of SEQ ID NO: 2 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1079-1291 of SEQ ID NO: 2; a modified BoNT/C1 H_(CC) binding domain comprising amino acids 1093-1291 of SEQ ID NO: 3 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1093-1291 of SEQ ID NO: 3; a modified BoNT/D H_(CC) binding domain comprising amino acids 1080-1276 of SEQ ID NO: 4 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1080-1276 of SEQ ID NO: 4; a modified BoNT/E H_(CC) binding domain comprising amino acids 1067-1252 of SEQ ID NO: 5 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1067-1252 of SEQ ID NO: 5; a modified BoNT/F H_(CC) binding domain comprising amino acids 1087-1274 of SEQ ID NO: 6 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1087-1274 of SEQ ID NO: 6; a modified BoNT/G H_(CC) binding domain comprising amino acids 1087-1297 of SEQ ID NO: 7 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1087-1297 of SEQ ID NO: 7; a modified TeNT H_(CC) binding domain comprising amino acids 1109-1315 of SEQ ID NO: 8 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1109-1315 of SEQ ID NO: 8, a modified BaNT H_(CC) binding domain comprising amino acids 1095-1269 of SEQ ID NO: 9 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1095-1268 of SEQ ID NO: 9, and a modified BuNT H_(CC) binding domain comprising amino acids 1086-1251 of SEQ ID NO: 10 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to the amino acid region 1086-1251 of SEQ ID NO: 10.

It is recognized by those of skill in the art that within each serotype of Clostridial toxin there can be naturally occurring Clostridial toxin binding domain variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, with specific binding domain subtypes showing approximately 87% amino acid identity when compared to another BoNT/A binding domain subtype. As used herein, the term “naturally occurring Clostridial toxin binding domain variant” means any Clostridial toxin binding domain produced by a naturally-occurring process, including, without limitation, Clostridial toxin binding domain isoforms produced from alternatively-spliced transcripts, Clostridial toxin binding domain isoforms produced by spontaneous mutation and Clostridial toxin binding domain subtypes. A naturally occurring Clostridial toxin binding domain variant can function in substantially the same manner as the reference Clostridial toxin binding domain on which the naturally occurring Clostridial toxin binding domain variant is based, and can be substituted for the reference Clostridial toxin binding domain in any aspect of the present invention. A naturally occurring Clostridial toxin binding domain variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference Clostridial toxin binding domain on which the naturally occurring Clostridial toxin binding domain variant is based. A naturally occurring Clostridial toxin binding domain variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial toxin binding domain on which the naturally occurring Clostridial toxin binding domain variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial toxin binding domain on which the naturally occurring Clostridial toxin binding domain variant is based.

A non-limiting examples of a naturally occurring Clostridial toxin binding domain variant is a Clostridial toxin binding domain isoform such as, e.g., a BoNT/A binding domain isoform, a BoNT/B binding domain isoform, a BoNT/C1 binding domain isoform, a BoNT/D binding domain isoform, a BoNT/E binding domain isoform, a BoNT/F binding domain isoform, a BoNT/G binding domain isoform, a TeNT binding domain isoform, a BaNT binding domain isoform, and a BuNT binding domain isoform. A Clostridial toxin binding domain isoform can function in substantially the same manner as the reference Clostridial toxin binding domain on which the Clostridial toxin binding domain isoform is based, and can be substituted for the reference Clostridial toxin binding domain in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxin binding domain variant is a Clostridial toxin binding domain subtype such as, e.g., a binding domain from subtype BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4; a binding domain from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B nonproteolytic; a binding domain from subtype BoNT/C1-1 and BoNT/C1-2; a binding domain from subtype BoNT/E1, BoNT/E2 and BoNT/E3; and a binding domain from subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4. A Clostridial toxin binding domain subtype can function in substantially the same manner as the reference Clostridial toxin binding domain on which the Clostridial toxin binding domain subtype is based, and can be substituted for the reference Clostridial toxin binding domain in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxin binding domain variant” means any Clostridial toxin binding domain produced with the aid of human manipulation, including, without limitation, Clostridial toxin binding domains produced by genetic engineering using random mutagenesis or rational design and Clostridial toxin binding domains produced by chemical synthesis. Non-limiting examples of non-naturally occurring Clostridial toxin binding domain variants include, e.g., conservative Clostridial toxin binding domain variants, non-conservative Clostridial toxin binding domain variants, Clostridial toxin binding domain chimeric variants and active Clostridial toxin binding domain fragments.

As used herein, the term “conservative Clostridial toxin binding domain variant” means a Clostridial toxin binding domain that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference Clostridial toxin binding domain sequence (Table 1). Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative Clostridial toxin binding domain variant can function in substantially the same manner as the reference Clostridial toxin binding domain on which the conservative Clostridial toxin binding domain variant is based, and can be substituted for the reference Clostridial toxin binding domain in any aspect of the present invention. A conservative Clostridial toxin binding domain variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, 400 or more amino acids, or 500 or more amino acids from the reference Clostridial toxin binding domain on which the conservative Clostridial toxin binding domain variant is based. A conservative Clostridial toxin binding domain variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial toxin binding domain on which the conservative Clostridial toxin binding domain variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial toxin binding domain on which the conservative Clostridial toxin binding domain variant is based. Non-limiting examples of a conservative Clostridial toxin binding domain variant include, e.g., conservative BoNT/A binding domain variants, conservative BoNT/B binding domain variants, conservative BoNT/C1 binding domain variants, conservative BoNT/D binding domain variants, conservative BoNT/E binding domain variants, conservative BoNT/F binding domain variants, conservative BoNT/G binding domain variants, conservative TeNT binding domain variants, conservative BaNT binding domain variants and conservative BuNT binding domain variants.

As used herein, the term “non-conservative Clostridial toxin binding domain variant” means a Clostridial toxin binding domain in which 1) at least one amino acid is deleted from the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based; 2) at least one amino acid added to the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain is based; or 3) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to that of the original amino acid from the reference Clostridial toxin binding domain sequence (Table 1). A non-conservative Clostridial toxin binding domain variant can function in substantially the same manner as the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based, and can be substituted for the reference Clostridial toxin binding domain in any aspect of the present invention. A non-conservative Clostridial toxin binding domain variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based. A non-conservative Clostridial toxin binding domain variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based. A non-conservative Clostridial toxin binding domain variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino acids, 400 or more amino acids, or 500 or more amino acids from the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based. A non-conservative Clostridial toxin binding domain variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial toxin binding domain on which the non-conservative Clostridial toxin binding domain variant is based. Non-limiting examples of a non-conservative Clostridial toxin binding domain variant include, e.g., non-conservative BoNT/A binding domain variants, non-conservative BoNT/B binding domain variants, non-conservative BoNT/C1 binding domain variants, non-conservative BoNT/D binding domain variants, non-conservative BoNT/E binding domain variants, non-conservative BoNT/F binding domain variants, non-conservative BoNT/G binding domain variants, non-conservative TeNT binding domain variants, non-conservative BaNT binding domain variants and non-conservative BuNT binding domain variants.

As used herein, the term “Clostridial toxin binding domain chimeric” means a polypeptide comprising at least a portion of a Clostridial toxin binding domain and at least a portion of at least one other polypeptide to form a toxin binding domain with at least one property different from the reference Clostridial toxin binding domains of Table 1, with the proviso that this Clostridial toxin binding domain chimeric is still capable of executing the cell binding step of the intoxication process, including, e.g., the selective binding of the Clostridial toxin to a toxin-specific receptor located on the plasma membrane surface of a target cell.

As used herein, the term “active Clostridial toxin binding domain fragment” means any of a variety of Clostridial toxin fragments comprising the binding domain can be useful in aspects of the present invention with the proviso that these active fragments can execute the cell binding step of the intoxication process, including, e.g., the selective binding of the Clostridial toxin to a toxin-specific receptor located on the plasma membrane surface of a target cell. For example, the H_(C) binding domains from the heavy chains of Clostridial toxins are approximately 400-435 amino acids in length and comprise a translocation domain (Table 1). Research has shown that the entire length of a binding domain from a Clostridial toxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include Clostridial toxin binding domains comprising a translocation domain having a length of, e.g., at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include Clostridial toxin binding domains comprising translocation domain having a length of, e.g., at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids. As another example, the H_(CC) binding domain from the heavy chains of Clostridial toxins are approximately 165-195 amino acids in length and comprise a binding domain (Table 1). Research has shown that the entire length of a H_(CC) binding domain from a Clostridial toxin heavy chain is not necessary for the binding activity of the binding domain. Thus, aspects of this embodiment can include a Clostridial toxin H_(CC) binding domain comprising a binding domain having a length of, e.g., at least 150 amino acids, at least 175 amino acids, at least 200 amino acids and at least 225 amino acids. Other aspects of this embodiment can include a Clostridial toxin H_(CC) binding domain comprising a binding domain having a length of, e.g., at most 150 amino acids, at most 175 amino acids, at most 200 amino acids and at most 225 amino acids.

Any of a variety of sequence alignment methods can be used to determine percent identity of naturally-occurring Clostridial toxin binding domain variants and non-naturally-occurring Clostridial toxin binding domain variants, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a multivalent Clostridial toxin disclosed in the present specification comprises a Clostridial toxin binding domain. In an aspect of this embodiment, a Clostridial toxin binding domain comprises a naturally occurring Clostridial toxin binding domain variant, such as, e.g., a Clostridial toxin binding domain isoform or a Clostridial toxin binding domain subtype. In another aspect of this embodiment, a Clostridial toxin binding domain comprises a non-naturally occurring Clostridial toxin binding domain variant, such as, e.g., a conservative Clostridial toxin binding domain variant, a non-conservative Clostridial toxin binding domain variant, a Clostridial toxin chimeric binding domain, an active Clostridial toxin binding domain fragment, or any combination thereof.

In an embodiment, a binding domain comprises a BoNT/A binding domain. In an aspect of this embodiment, a BoNT/A binding domain comprises a BoNT/A H_(C) binding domain. In aspects of this embodiment, a BoNT/A binding domain comprising BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at least 75% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at least 80% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at least 85% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at least 90% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1 or at least 95% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at most 75% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at most 80% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at most 85% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at most 90% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1 or at most 95% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 874-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 874-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 874-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 874-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 874-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 874-1296 of SEQ ID NO: 1.

In another aspect of this embodiment, a BoNT/A binding domain comprises a BoNT/A H_(CC) binding domain. In aspects of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC) binding domain comprises a modification of amino acids 1111-1296 of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/A H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/A H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/A H_(CC) binding domain. In another aspect of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC) binding domain comprises a modification to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 75% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 80% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 85% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 90% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1 or at least 95% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most 75% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most 80% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most 85% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most 90% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1 or at most 95% amino acid identity with amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In another embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A H_(CC) binding domain. In another aspect of this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC) binding domain comprises a modification of amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 75% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 80% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 85% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 90% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 or at least 95% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at most 75% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at most 80% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at most 85% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at most 90% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 or at most 95% amino acid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprises a substitution of amino acid Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270, Gly 1279 or Trp 1282, or any combination thereof, the substitution enhancing the binding activity of the BoNT/A H_(CC) binding domain. In other aspects of this embodiment, a BoNT/A H_(CC) binding domain comprises a deletion of amino acid Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270, Gly 1279 or Trp 1282, or any combination thereof, the deletion enhancing the binding activity of the BoNT/A H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/B binding domain. In an aspect of this embodiment, a BoNT/B binding domain comprises a BoNT/B H_(C) binding domain. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide, e.g., at least 70% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at least 75% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at least 80% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at least 85% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at least 90% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2 or at least 95% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at most 75% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at most 80% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at most 85% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at most 90% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2 or at most 95% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 861-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 861-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 861-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 861-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 861-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 861-1291 of SEQ ID NO: 2.

In another aspect of this embodiment, a BoNT/B binding domain comprises a BoNT/B H_(CC) binding domain. In another aspect of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(CC) binding domain comprises an α-fold motif of a β-trefoil domain of a BoNT/B H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/B H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/B H_(CC) binding domain. In another aspect of this embodiment, a BoNT/B binding domain comprising a BoNT/B H_(CC) binding domain comprises a modification to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 75% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 80% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 85% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 90% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2 or at least 95% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most 75% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most 80% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most 85% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most 90% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2 or at most 95% amino acid identity with amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In another embodiment, a binding domain comprising a BoNT/B H binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B H_(CC) binding domain. In another aspect of this embodiment, a binding domain comprising a BoNT/B H_(CC) binding domain comprises a modification of amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 75% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 80% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 85% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 90% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 or at least 95% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at most 75% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at most 80% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at most 85% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at most 90% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 or at most 95% amino acid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprises a substitution of amino acid Trp 1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142, Ile 1147, Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263, Glu 1266, Gly 1277 or Trp 1280, or any combination thereof, the substitution enhancing the binding capability of the BoNT/B H_(CC) binding domain. In other aspects of this embodiment, a BoNT/B H_(CC) binding domain comprises a deletion of amino acid Trp 1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142, Ile 1147, Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263, Glu 1266, Gly 1277 or Trp 1280, or any combination thereof, the deletion enhancing the binding capability of the BoNT/B H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/C1 binding domain. In an aspect of this embodiment, a BoNT/C1 binding domain comprises a BoNT/C1 H_(C) binding domain. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at least 75% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at least 80% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at least 85% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at least 90% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3 or at least 95% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at most 75% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at most 80% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at most 85% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at most 90% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3 or at most 95% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 869-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 869-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 869-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 869-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 869-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 869-1291 of SEQ ID NO: 3.

In another aspect of this embodiment, a BoNT/C1 binding domain comprises a BoNT/C1 H_(CC) binding domain. In an aspect of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domain comprises a modification of amino acids 1112-1291 of SEQ ID NO: 3. In another aspect of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/C1 H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/C1 H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/C1 H_(CC) binding domain. In another aspect of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domain comprises a modification to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 75% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 80% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 85% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 90% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3 or at least 95% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most 75% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most 80% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most 85% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most 90% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3 or at most 95% amino acid identity with amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In another embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 H_(CC) binding domain. In another aspect of this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domain comprises a modification of amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 75% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 80% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 85% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 90% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 or at least 95% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at most 75% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at most 80% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at most 85% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at most 90% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 or at most 95% amino acid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprises a substitution of amino acid Trp 1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145, Ile 1150, Asp 1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259, His 1261, Gly 1281 or Trp 1284, or any combination thereof, the substitution enhancing the binding activity of the BoNT/C1 H_(CC) binding domain. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprises a deletion of amino acid Trp 1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145, Ile 1150, Asp 1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259, His 1261, Gly 1281 or Trp 1284, or any combination thereof, the deletion enhancing the binding activity of the BoNT/C1 H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/D binding domain. In an aspect of this embodiment, a BoNT/D binding domain comprises a BoNT/D H_(C) binding domain. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at least 75% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at least 80% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at least 90% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4 or at least 95% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at most 75% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at most 80% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at most 85% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at most 90% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4 or at most 95% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 865-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 865-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 865-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 865-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 865-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 865-1276 of SEQ ID NO: 4.

In another aspect of this embodiment, a BoNT/D binding domain comprises a BoNT/D H_(CC) binding domain. In an aspect of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC) binding domain comprises a modification of amino acids 1099-1276 of SEQ ID NO: 4. In another aspect of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/D H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/D H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/D H_(CC) binding domain. In another aspect of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC) binding domain comprises a modification to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 75% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 80% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 90% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4 or at least 95% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most 75% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most 80% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most 85% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most 90% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4 or at most 95% amino acid identity with amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In another embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/D H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D H_(CC) binding domain. In another aspect of this embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC) binding domain comprises a modification of amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least 75% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least 80% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least 90% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 or at least 95% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 75% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 80% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 85% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 90% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 or at most 95% amino acid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprises a substitution of amino acid Trp 1089, Gly 1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile 1137, Asp 1153, Asn 1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser 1262 or Trp 1265, or any combination thereof, the substitution enhancing the binding activity of the BoNT/D H_(CC) binding domain. In other aspects of this embodiment, a BoNT/D H_(CC) binding domain comprises a deletion of amino acid Trp 1089, Gly 1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile 1137, Asp 1153, Asn 1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser 1262 or Trp 1265, or any combination thereof, the deletion enhancing the binding activity of the BoNT/D H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/E binding domain. In an aspect of this embodiment, a BoNT/E binding domain comprises a BoNT/E H_(C) binding domain. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at least 75% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at least 80% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at least 85% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at least 90% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5 or at least 95% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at most 75% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at most 80% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at most 85% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at most 90% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5 or at most 95% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 848-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 848-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 848-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 848-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 848-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 848-1252 of SEQ ID NO: 5.

In another aspect of this embodiment, a BoNT/E binding domain comprises a BoNT/E H_(CC) binding domain. In an aspect of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC) binding domain comprises a modification of amino acids 1086-1252 of SEQ ID NO: 5. In another aspect of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/E H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/E H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/E H_(CC) binding domain. In another aspect of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC) binding domain comprises a modification to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 75% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 80% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 85% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 90% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5 or at least 95% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most 75% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most 80% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most 85% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most 90% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5 or at most 95% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In another embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/E H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/E H_(CC) binding domain. In another aspect of this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC) binding domain comprises a modification of amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least 75% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least 80% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least 85% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least 90% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 or at least 95% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 75% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 80% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 85% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 90% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 or at most 95% amino acid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprises a substitution of amino acid Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or any combination thereof, the substitution enhancing the binding activity of the BoNT/E H_(CC) binding domain. In other aspects of this embodiment, a BoNT/E H_(CC) binding domain comprises a deletion of amino acid Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or any combination thereof, the deletion enhancing the binding activity of the BoNT/E H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/F binding domain. In an aspect of this embodiment, a BoNT/F binding domain comprises a BoNT/F H_(C) binding domain. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at least 75% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at least 80% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at least 85% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at least 90% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6 or at least 95% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at most 75% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at most 80% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at most 85% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at most 90% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6 or at most 95% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 867-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 867-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 867-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 867-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 867-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 867-1274 of SEQ ID NO: 6.

In another aspect of this embodiment, a BoNT/F binding domain comprises a BoNT/F H_(CC) binding domain. In an aspect of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC) binding domain comprises a modification of amino acids 1106-1274 of SEQ ID NO: 6. In another aspect of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/F H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/F H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/F H_(CC) binding domain. In another aspect of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC) binding domain comprises a modification to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 75% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 80% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 85% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 90% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6 or at least 95% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most 75% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most 80% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most 85% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most 90% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6 or at most 95% amino acid identity with amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In another embodiment, a BoNT/F binding domain comprises a BoNT/F H_(CC) binding domain of comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/F H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/F H_(CC) binding domain. In another aspect of this embodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC) binding domain comprises a modification of amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 75% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 80% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 85% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 90% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 or at least 95% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at most 75% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at most 80% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at most 85% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at most 90% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 or at most 95% amino acid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprises a substitution of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147, Ile 1152, Asp 1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248, Asn 1251, Gly 1260 or Trp 1263, or any combination thereof, the substitution enhancing the binding activity of the BoNT/F H_(CC) binding domain. In other aspects of this embodiment, a BoNT/F H_(CC) binding domain comprises a deletion of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147, Ile 1152, Asp 1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248, Asn 1251, Gly 1260 or Trp 1263, or any combination thereof, the deletion enhancing the binding activity of the BoNT/F H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/G binding domain. In an aspect of this embodiment, a BoNT/G binding domain comprises a BoNT/G H_(C) binding domain. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at least 75% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at least 80% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at least 85% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at least 90% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7 or at least 95% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at most 75% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at most 80% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at most 85% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at most 90% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7 or at most 95% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 866-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 866-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 866-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 866-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 866-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 866-1297 of SEQ ID NO: 7.

In another aspect of this embodiment, a BoNT/G binding domain comprises a BoNT/G H_(CC) binding domain. In an aspect of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC) binding domain comprises a modification of amino acids 1106-1297 of SEQ ID NO: 7. In another aspect of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BoNT/G H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BoNT/G H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/G H_(CC) binding domain. In another aspect of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC) binding domain comprises a modification to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 75% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 80% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 85% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 90% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7 or at least 95% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most 75% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most 80% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most 85% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most 90% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7 or at most 95% amino acid identity with amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In another embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/G H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/G H_(CC) binding domain. In another aspect of this embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC) binding domain comprises a modification of amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 75% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 80% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 85% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 90% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 or at least 95% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at most 75% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at most 80% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at most 85% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at most 90% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 or at most 95% amino acid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprises a substitution of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148, Ile 1153, Asp 1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269, Arg 1272, Gly 1283 or Trp 1285, or any combination thereof, the substitution enhancing the binding activity of the BoNT/G H_(CC) binding domain. In other aspects of this embodiment, a BoNT/G H_(CC) binding domain comprises a deletion of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148, Ile 1153, Asp 1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269, Arg 1272, Gly 1283 or Trp 1285, or any combination thereof, the deletion enhancing the binding activity of the BoNT/G H_(CC) binding domain.

In another embodiment, a binding domain comprises a TeNT binding domain. In an aspect of this embodiment, a TeNT binding domain comprises a TeNT H_(C) binding domain. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at least 75% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at least 80% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at least 90% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8 or at least 95% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at most 75% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at most 80% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at most 85% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, at most 90% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8 or at most 95% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 882-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 882-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide e having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 882-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 882-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 882-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT binding domain comprising a TeNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 882-1315 of SEQ ID NO: 8.

In another aspect of this embodiment, a TeNT binding domain comprises a TeNT H_(CC) binding domain. In an aspect of this embodiment, a TeNT binding domain comprising a TeNT H_(CC) binding domain comprises a modification of amino acids 1128-1315 of SEQ ID NO: 8. In another aspect of this embodiment, a TeNT H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a TeNT H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a TeNT H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a TeNT H_(CC) binding domain. In another aspect of this embodiment, a TeNT binding domain comprising a TeNT H_(CC) binding domain comprises a modification to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 75% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 80% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 90% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8 or at least 95% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most 75% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most 80% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most 85% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most 90% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8 or at most 95% amino acid identity with amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In another embodiment, a TeNT binding domain comprising a TeNT H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a TeNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a TeNT H_(CC) binding domain. In another aspect of this embodiment, a TeNT binding domain comprising a TeNT H_(CC) binding domain comprises a modification of amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 75% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 80% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 90% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 or at least 95% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at most 75% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at most 80% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at most 85% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at most 90% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 or at most 95% amino acid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replaced with phenylalanine. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replaced with phenylalanine. In yet other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domain comprises a substitution of amino acid Trp 1118, Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172, Ile 1177, Asp 1194, Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290, His 1293, Gly 1300 or Trp 1303, or any combination thereof, the substitution enhancing the binding activity of the TeNT H_(CC) binding domain. In other aspects of this embodiment, a TeNT H_(CC) binding domain comprises a deletion of amino acid Trp 1118, Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172, Ile 1177, Asp 1194, Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290, His 1293, Gly 1300 or Trp 1303, or any combination thereof, the deletion enhancing the binding activity of the TeNT H_(CC) binding domain.

In another embodiment, a binding domain comprises a BaNT binding domain. In an aspect of this embodiment, a BaNT binding domain comprises a BaNT H_(C) binding domain. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at least 75% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at least 80% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at least 90% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9 or at least 95% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at most 75% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at most 80% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at most 85% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, at most 90% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9 or at most 95% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 858-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 858-1268 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 858-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 858-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 858-1268 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT binding domain comprising a BaNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 858-1268 of SEQ ID NO: 9.

In another embodiment, a BaNT binding domain comprises a BaNT H_(CC) binding domain. In an aspect of this embodiment, a BaNT binding domain comprising a BaNT H_(CC) binding domain comprises a modification of amino acids 1128-1315 of SEQ ID NO: 8. In another aspect of this embodiment, a BaNT binding domain comprising a BaNT H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BaNT H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BaNT H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BaNT H_(CC) binding domain. In another aspect of this embodiment, a BaNT binding domain comprising a BaNT H_(CC) binding domain comprises a modification to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at least 75% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at least 80% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at least 90% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9 or at least 95% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at most 75% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at most 80% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at most 85% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at most 90% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9 or at most 95% amino acid identity with amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In another embodiment, a BaNT binding domain comprising a BaNT H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BaNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BaNT H_(CC) binding domain. In another aspect of this embodiment, a BaNT binding domain comprising a BaNT H_(CC) binding domain comprises a modification of amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least 75% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least 80% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least 90% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 or at least 95% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at most 75% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at most 80% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at most 85% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at most 90% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 or at most 95% amino acid identity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In still other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domain comprises a substitution of amino acid Trp 1085, Gly 1086, Leu 1089, Tyr 1095, Tyr 1096, Gly 1137, Ile 1142, Asp 1161, Ile 1189, Phe 1231, Ser 1239, Trp 1241, Tyr 1242, Asn 1245, Gly 1254 or Trp 1257, or any combination thereof, the substitution enhancing the binding activity of the BaNT H_(CC) binding domain. In other aspects of this embodiment, a BaNT H_(CC) binding domain comprises a deletion of amino acid Trp 1085, Gly 1086, Leu 1089, Tyr 1095, Tyr 1096, Gly 1137, Ile 1142, Asp 1161, Ile 1189, Phe 1231, Ser 1239, Trp 1241, Tyr 1242, Asn 1245, Gly 1254 or Trp 1257, or any combination thereof, the deletion enhancing the binding activity of the BaNT H_(CC) binding domain.

In another embodiment, a binding domain comprises a BuNT binding domain. In an aspect of this embodiment, a BuNT binding domain comprises a BuNT H_(C) binding domain. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at least 75% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at least 80% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at least 85% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at least 90% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10 or at least 95% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at most 75% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at most 80% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at most 85% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, at most 90% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10 or at most 95% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous amino acid substitutions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutions relative to amino acids 848-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 848-1251 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relative to amino acids 848-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 848-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids 848-1251 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT binding domain comprising a BuNT H_(C) binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additions relative to amino acids 848-1251 of SEQ ID NO: 10.

In another embodiment, a BuNT binding domain comprises a BuNT H_(CC) binding domain. In an aspect of this embodiment, a BuNT binding domain comprising a BuNT H_(CC) binding domain comprises a modification of amino acids 1128-1315 of SEQ ID NO: 8. In another aspect of this embodiment, a BuNT binding domain comprising a BuNT H_(CC) binding domain comprises a α-fold motif of a β-trefoil domain of a BuNT H_(CC) binding domain, a β-fold motif of a β-trefoil domain of a BuNT H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain of a BuNT H_(CC) binding domain. In another aspect of this embodiment, a BuNT binding domain comprising a BuNT H_(CC) binding domain comprises a modification to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at least 75% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at least 80% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at least 85% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at least 90% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10 or at least 95% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at most 75% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at most 80% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at most 85% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at most 90% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10 or at most 95% amino acid identity with amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In another embodiment, a BuNT binding domain comprising a BuNT H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BuNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of a BuNT H_(CC) binding domain. In another aspect of this embodiment, a BuNT binding domain comprising a BuNT H_(CC) binding domain comprises a modification of amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at least 75% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at least 80% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at least 85% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at least 90% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 or at least 95% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at most 75% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at most 80% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at most 85% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at most 90% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 or at most 95% amino acid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 can be replaced with phenylalanine. In yet other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprising a β-trefoil fold domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid additions relative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domain comprises a substitution of amino acid Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or any combination thereof, the substitution enhancing the binding activity of the BuNT H_(CC) binding domain. In other aspects of this embodiment, a BuNT H_(CC) binding domain comprises a deletion of amino acid Trp 1085, Gly 1086, Leu 1089, Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or any combination thereof, the deletion enhancing the binding activity of the BuNT H_(CC) binding domain.

Another example of a binding domain, includes, without limitation, a Clostridial toxin non-toxin associated protein, such as, e.g., non-toxic non-hemagglutinin (NTNH), hemagglutinin-17 (HA-17), hemagglutinin-33 (HA-33) and hemagglutinin-70 (HA-70). In vivo, Clostridial bacteria produce a toxin complex comprising the approximately 150-kDa Clostridial toxin and other proteins collectively called nontoxin associated proteins (NAPs). Identified NAPs include proteins possessing hemaglutination activity, such, e.g., a hemagglutinin of approximately 17-kDa (HA-17), a hemagglutinin of approximately 33-kDa (HA-33) and a hemagglutinin of approximately 70-kDa (HA-70); as well as non-toxic non-hemagglutinin (NTNH), a protein of approximately 130-kDa, see, e.g., Eric A. Johnson and Marite Bradshaw, Clostridial botulinum and its Neurotoxins: A Metabolic and Cellular Perspective, 39 Toxicon 1703-1722 (2001); and Stephanie Raffestin et al., Organization and Regulation of the Neurotoxin Genes in Clostridium botulinum and Clostridium tetani, 10 Anaerobe 93-100 (2004). The toxin complex is important for the intoxication process because it provides protection from adverse environmental conditions, resistance to protease digestion, and appears to facilitate internalization and activation of the toxin.

Recent crystallography experiments have revealed that HA-17, HA-33 and NTNH from various Clostridial bacteria contain a region comprising β-trefoil domains very similar to the single β-trefoil domain present in the binding domain of Clostridial toxins, see, e.g., Kaoru Inoue et al., Structural Analysis by X-Ray Crystallography and Calorimetry of a Haemagglutinin Component (HA 1) of the Progenitor Toxin from Clostridium botulinum, 149 Microbiol. 3361-3370 (2003); and Joseph W. Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/A from Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in the Progenitor Toxin Complex, 346 J. Mol. Biol. 1083-1093 (2005). For example, HA-33 from Clostridium botulinum serotype A has two β-trefoil domains, each of which consists of three potential carbohydrate binding moieties or β-trefoil folds, designated 1α (amino acids 10-55 of SEQ ID NO: 11), 1β (amino acids 56-102 of SEQ ID NO: 11) and 1γ (amino acids 103-144 of SEQ ID NO: 11) for the first β-trefoil domain and 2α (amino acids 151-197 of SEQ ID NO: 11), 2β (amino acids 198-245 of SEQ ID NO: 11) and 2γ (amino acids 246-293 of SEQ ID NO: 11) for the second β-trefoil domain. Mutations in conserved amino acids of the carbohydrate binding moiety result in a loss of carbohydrate binding, see, e.g., Kaoru Inoue et al., Structural Analysis by X-Ray Crystallography and Calorimetry of a Haemagglutinin Component (HA1) of the Progenitor Toxin from Clostridium botulinum, 149 Microbiol. 3361-3370 (2003).

TABLE 3 β-trefoil Domains of Clostridial HA-33 Proteins Amino Acid Sequence Region of Carbohydrate Binding Moieties 1β4/β5 1β8/β9 Protein SEQ ID NO: 1α-fold β-hairpin turn 1β-fold β-hairpin turn 1γ-fold HA-33/A1 11 10-54 55-59 60-100 101-104 105-144 HA-33/A2 12 10-54 55-59 60-100 101-104 105-144 HA-33/A3 13 10-54 55-59 60-100 101-104 105-144 HA-33/A4 14 10-56 57-61 62-102 103-106 107-146 HA-33/A5 15 10-54 55-59 60-100 101-104 105-144 HA-33/B1 16 10-54 55-59 60-100 101-104 105-144 HA-33/B2 17 10-56 57-61 62-102 103-106 107-146 HA-33/C1-1 18 10-54 55-59 60-98   99-102 103-141 HA-33/C1-2 19 10-54 55-59 60-98   99-102 103-141 HA-33/D1 20 10-54 55-59 60-98   99-102 103-141

These β-trefoil domains are also found in the HA-33 proteins produced by Clostridium botulinum serotype B, serotype C1 and serotype D and sequence alignments revealed that amino acids essential for overall carbohydrate binding are conserved. The amino acids predicted to be essential for carbohydrate binding are as follows Asp 263, Tyr 265, Gln 268, Gln 276, Phe 278 and Gln 286 of HA-33/A1 of SEQ ID NO: 11, HA-33/A2 of SEQ ID NO: 12, HA-33/A3 of SEQ ID NO: 13, HA-33/A5 of SEQ ID NO: 14 and HA-33/B2 of SEQ ID NO: 15; Asp 264, Tyr 266, Gln 269, Gln 277, Phe 279 and Gln 287 of HA-33/A4 of SEQ ID NO: 14; Asp 262, Tyr 264, Gln 267, Gln 275, Phe 277 and Gln 285 of HA-33/B1 of SEQ ID NO: 16; Asp 255, Val 257, Gly 260, Gln 268, Typ 270 and Gln 278 of HA-33/C1 of SEQ ID NO: 18; and Asp 256, Tyr 258, Gln 261, Ile 269, Asp 271 and Gln 279 of HA-33/C2 of SEQ ID NO: 19 and HA-33/D of SEQ ID NO: 20.

Immunoaffinity column chromatography and pull-down assays have shown that HA-33 can bind synaptotagmin II, a putative Clostridial toxin receptor, see, e.g., Yu Zhou et al., Haemagglutinin-33 of Type Q Botulinum Neurotoxin Complex Binds with Synaptotagmin II, 272 FEBS Lett. 2717-2726 (2005). The amino acid sequences comprising the β-trefoil domains found in various Clostridial HA-33 proteins are shown in Tables 3 and 4.

TABLE 4 β-trefoil Domains of Clostridial HA-33 Proteins Amino Acid Sequence Region of Carbohydrate Binding Moieties 2β4/β5 2β8/β9 Protein SEQ ID NO: 2α-fold β-hairpin turn 2β-fold β-hairpin turn 2γ-fold HA-33/A1 11 151-195 196-199 200-242 243-248 249-293 HA-33/A2 12 151-195 196-199 200-242 243-248 249-293 HA-33/A3 13 151-195 196-199 200-242 243-248 249-293 HA-33/A4 14 153-197 198-201 202-243 244-249 250-294 HA-33/A5 15 151-195 196-199 200-242 243-248 249-279 HA-33/B1 16 151-195 196-199 200-241 242-247 248-292 HA-33/B2 17 153-197 198-201 200-242 243-248 249-291 HA-33/C1-1 18 148-190 191-194 195-234 235-240 241-285 HA-33/C1-2 19 148-190 191-194 195-235 236-241 242-286 HA-33/D1 20 148-190 191-194 195-235 236-241 242-286

Further analysis of the β-trefoil domain sequence of HA-33 also identified β-trefoil domains in HA-17 and NTNH, see, e.g., Joseph W. Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/A from Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in the Progenitor Toxin Complex, 346 J. Mol. Biol. 1083-1093 (2005). The HA-17 comprises a single β-trefoil domain containing three carbohydrate binding moieties or β-trefoil folds. The carbohydrate binding moieties of HA-17 exhibits the greatest sequence similarity with the 2γ carbohydrate binding moiety of HA-33. These R-trefoil domains are also found in the HA-17 proteins produced by Clostridium botulinum serotype B, serotype C1 and serotype D and sequence alignments revealed that amino acids essential for overall carbohydrate binding are conserved. The amino acids predicted to be essential for carbohydrate binding are as follows Tyr 110, Typ 112, Tyr 115, Pro 130, Phe 132 and Asn 138 of HA-17/A of SEQ ID NO: 21, HA-17/B of SEQ ID NO: 22, HA-17/C1 of SEQ ID NO: 23 and HA-17/D of SEQ ID NO: 24. The amino acid sequences comprising the β-trefoil domains found in various Clostridial HA-17 proteins are shown in Table 5.

TABLE 5 β-trefoil Domains of Clostridial HA-17 Proteins Amino Acid Sequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold HA-17/A 21 9-50 51-54 55-91 92-94 95-146 HA-17/B 22 9-50 51-54 55-91 92-94 95-146 HA-17/C1 23 9-50 51-54 55-91 92-94 95-146 HA-17/D 24 9-50 51-54 55-91 92-94 95-146

NTNH from various Clostridial bacteria shows significant sequence similarity to the β-trefoil domains present in the cell binding domain of BoNT/A and TeNT. The high degree of structural similarity is interesting in light of the low sequence similarity between NTNH and the Clostridial toxins. Furthermore, since NTNH of the various serotypes have greater sequence similarity than the Clostridial toxins, it is likely that the NTNH produced by other Clostridial strains will also have β-trefoil domains exhibiting high structural similarity with the binding domains of Clostridial toxins. The β-trefoil domains of various Clostridial NTNHs are as follows: amino acids 1050-1193 of NTNH/A1 of SEQ ID NO: 25; amino acids 1050-1198 of NTNH/A2 of SEQ ID NO: 26; amino acids 1050-1193 of NTNH/A3 of SEQ ID NO: 27; amino acids 1049-1197 of NTNH/B of SEQ ID NO: 28; amino acids 1049-1196 of NTNH/C1 of SEQ ID NO: 29; amino acids 1049-1196 of NTNH/D of SEQ ID NO: 30; amino acids 1014-1162 of NTNH/E of SEQ ID NO: 30; amino acids 1016-1159 of NTNH/F1 of SEQ ID NO: 32; amino acids 1017-1165 of NTNH/F2 of SEQ ID NO: 33; and amino acids 1050-1198 of NTNH/G of SEQ ID NO: 34. The amino acid sequences comprising the β-trefoil domains found in various Clostridial NTNH proteins are shown in Table 6.

TABLE 6 β-trefoil Domains of Clostridial NTNH Proteins Amino Acid Sequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold NTNH/A1 25 1050-1097 1098-1110 1111-1138 1139-1148 1149-1194 NTNH/A2 26 1050-1097 1098-1110 1111-1139 1140-1148 1149-1199 NTNH/A3 27 1050-1097 1098-1110 1111-1138 1139-1148 1149-1194 NTNH/B 28 1049-1096 1097-1109 1110-1138 1139-1147 1148-1198 NTNH/C1 29 1049-1096 1097-1109 1110-1138 1139-1147 1148-1197 NTNH/D 30 1049-1096 1097-1109 1110-1138 1139-1147 1148-1197 NTNH/E 31 1014-1061 1062-1074 1075-1103 1104-1113 1114-1163 NTNH/F1 32 1016-1063 1064-1076 1077-1104 1105-1114 1115-1160 NTNH/F2 33 1017-1064 1065-1077 1078-1106 1107-1116 1117-1166 NTNH/G 34 1050-1097 1098-1110 1111-1139 1140-1149 1150-1199

The β-trefoil domains present in the Clostridial toxin, HA-33, HA-17 and NTNH collectively form nearly half the mass of a Clostridial toxin complex and underlies the apparent importance of carbohydrate binding in the cell binding step of the intoxication process. This observation is further enhanced by the fact that a Clostridial toxin alone is not as effective in intoxicating a cell as the entire toxin complex. One potential explanation for this enhanced binding activity is the presence, both in type and in quantity, of the β-trefoil domains present in HA-33, HA-17 and NTNH. Therefore, the high prediction of structural similarity of the β-trefoil domains present in HA-33, HA-17 and NTNH relative to the β-trefoil domain found in Clostridial toxins provides a potential source of binding domains useful for developing multivalent Clostridial toxins. As a non-limiting example, a carbohydrate binding moiety or a β-trefoil fold from HA-33, HA-17 or NTNH can be substituted for the naturally occurring carbohydrate binding moiety present in a Clostridial toxin. As another non-limiting example, a carbohydrate binding moiety or a β-trefoil fold from HA-33, HA-17 or NTNH can be added in addition to the naturally occurring carbohydrate binding moiety present in a Clostridial toxin. As yet another non-limiting example, a multiple carbohydrate binding moieties or a β-trefoil folds from HA-33, HA-17 or NTNH can be substituted for the naturally occurring carbohydrate binding moiety present in a Clostridial toxin. As still another non-limiting example, multiple carbohydrate binding moieties or a β-trefoil folds from HA-33, HA-17 or NTNH can be added in addition to the naturally occurring carbohydrate binding moiety present in a Clostridial toxin. As another non-limiting example, multiple carbohydrate binding moieties or a β-trefoil folds from a Clostridial toxin binding domain can be added in addition to the naturally occurring carbohydrate binding moiety present in a Clostridial toxin.

As used herein, the term “Non-toxin Associated Protein” is synonymous with “NAP” and means a Clostridial NAP with selective binding activity, such as, e.g., a binding affinity or a binding specificity, for a Clostridial toxin receptor. It is envisioned that both naturally occurring NAPs as well as non-naturally occurring NAPs can be used as a binding domain. A Clostridial NAP includes, without limitation, naturally occurring Clostridial NAP variants, such as, e.g., Clostridial NAP isoforms and Clostridial NAP subtypes; non-naturally occurring Clostridial NAP variants, such as, e.g., conservative Clostridial NAP variants, non-conservative Clostridial NAP variants, Clostridial NAP chimerics, active Clostridial NAP fragments thereof, or any combination thereof.

As used herein, the term “Clostridial NAP variant,” whether naturally-occurring or non-naturally-occurring, means a Clostridial NAP that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Tables 3-6) and can be described in percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all Clostridial NAP variants disclosed in the present specification are capable of executing the cell binding step of the intoxication process.

It is recognized by those of skill in the art that within each Clostridial bacterium there can be naturally occurring Clostridial NAP variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently five BoNT/A HA-33 variants, HA-33/A1, HA-33/A2, HA-33/A3, HA-33/A4 and HA-33/A5 (Tables 3 and 4), with specific HA-33 variants showing various degrees of amino acid divergence when compared to another HA-33 variant. As another example, there are presently three BoNT/A NTNH-33 variants, NTNH/A1, NTNH/A2 and NTNH/A3 (Table 6), with specific NTNH variant showing various degrees of amino acid divergence when compared to another NTNH variant. As used herein, the term “naturally occurring Clostridial NAP variant” means any Clostridial NAP produced by a naturally-occurring process, including, without limitation, Clostridial NAP isoforms produced from alternatively-spliced transcripts, Clostridial NAP isoforms produced by spontaneous mutation and Clostridial NAP subtypes. A naturally occurring Clostridial NAP variant can function in substantially the same manner as the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention. A naturally occurring Clostridial NAP variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based. A naturally occurring Clostridial NAP variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based.

A non-limiting examples of a naturally occurring Clostridial NAP variant is a Clostridial NAP isoform such as, e.g., a BoNT/A HA-33 isoform, a Clostridium botulinum serotype B HA-33 isoform, a Clostridium botulinum serotype C1 HA-33 isoform, a Clostridium botulinum serotype D HA-33 isoform, a BoNT/A HA-17 isoform, a Clostridium botulinum serotype B HA-17 isoform, a Clostridium botulinum serotype C1 HA-17 isoform, a Clostridium botulinum serotype D HA-17 isoform, a BoNT/A NTNH isoform, a Clostridium botulinum serotype B NTNH isoform, a Clostridium botulinum serotype C1 NTNH isoform, a Clostridium botulinum serotype D NTNH isoform, a Clostridium botulinum serotype E NTNH isoform, a Clostridium botulinum serotype F NTNH isoform and a Clostridium botulinum serotype G NTNH isoform. A Clostridial NAP isoform can function in substantially the same manner as the reference Clostridial NAP on which the Clostridial NAP isoform is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial NAP variant is a Clostridial NAP subtype such as, e.g., a BoNT/A HA-33 subtype, a Clostridium botulinum serotype B HA-33 subtype, a Clostridium botulinum serotype C1 HA-33 subtype, a Clostridium botulinum serotype D HA-33 subtype, a BoNT/A HA-17 subtype, a Clostridium botulinum serotype B HA-17 subtype, a Clostridium botulinum serotype C1 HA-17 subtype, a Clostridium botulinum serotype D HA-17 subtype, a BoNT/A NTNH subtype, a Clostridium botulinum serotype B NTNH subtype, a Clostridium botulinum serotype C1 NTNH subtype, a Clostridium botulinum serotype D NTNH subtype, a Clostridium botulinum serotype E NTNH subtype, a Clostridium botulinum serotype F NTNH subtype and a Clostridium botulinum serotype G NTNH subtype. A Clostridial NAP subtype can function in substantially the same manner as the reference Clostridial NAP on which the Clostridial NAP subtype is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial NAP variant” means any Clostridial NAP produced with the aid of human manipulation, including, without limitation, Clostridial NAPs produced by genetic engineering using random mutagenesis or rational design and Clostridial NAPs produced by chemical synthesis. Non-limiting examples of non-naturally occurring Clostridial NAP variants include, e.g., conservative Clostridial NAP variants, non-conservative Clostridial NAP variants, Clostridial NAP chimeric variants and active Clostridial NAP fragments.

As used herein, the term “conservative Clostridial NAP variant” means a Clostridial NAP that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference Clostridial NAP sequence (see Tables 3-6). Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative Clostridial NAP variant can function in substantially the same manner as the reference Clostridial NAP on which the conservative Clostridial NAP variant is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention. A conservative Clostridial NAP variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids from the reference Clostridial NAP on which the conservative Clostridial NAP variant is based. A conservative Clostridial NAP variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial NAP on which the conservative Clostridial NAP variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial NAP on which the conservative Clostridial NAP variant is based. Non-limiting examples of a conservative Clostridial NAP variant include, e.g., a conservative BoNT/A HA-33 variant, a conservative Clostridium botulinum serotype B HA-33 variant, a conservative Clostridium botulinum serotype C1 HA-33 variant, a conservative Clostridium botulinum serotype D HA-33 variant, a conservative BoNT/A HA-17 variant, a conservative Clostridium botulinum serotype B HA-17 variant, a conservative Clostridium botulinum serotype C1 HA-17 variant, a conservative Clostridium botulinum serotype D HA-17 variant, a conservative BoNT/A NTNH variant, a conservative Clostridium botulinum serotype B NTNH variant, a conservative Clostridium botulinum serotype C1 NTNH variant, a conservative Clostridium botulinum serotype D NTNH variant, a conservative Clostridium botulinum serotype E NTNH variant, a conservative Clostridium botulinum serotype F NTNH variant and a conservative Clostridium botulinum serotype G NTNH variant.

As used herein, the term “non-conservative Clostridial NAP variant” means a Clostridial NAP in which 1) at least one amino acid is deleted from the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based; 2) at least one amino acid added to the reference Clostridial NAP on which the non-conservative Clostridial NAP is based; or 3) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to that of the original amino acid from the reference Clostridial NAP sequence (see Tables 3-6). A non-conservative Clostridial NAP variant can function in substantially the same manner as the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention. A non-conservative Clostridial NAP variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based. A non-conservative Clostridial NAP variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based. A non-conservative Clostridial NAP variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids from the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based. A non-conservative Clostridial NAP variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial NAP on which the non-conservative Clostridial NAP variant is based. Non-limiting examples of a non-conservative Clostridial NAP variant include, e.g., a non-conservative BoNT/A HA-33 variant, a non-conservative Clostridium botulinum serotype B HA-33 variant, a non-conservative Clostridium botulinum serotype C1 HA-33 variant, a non-conservative Clostridium botulinum serotype D HA-33 variant, a non-conservative BoNT/A HA-17 variant, a non-conservative Clostridium botulinum serotype B HA-17 variant, a non-conservative Clostridium botulinum serotype C1 HA-17 variant, a non-conservative Clostridium botulinum serotype D HA-17 variant, a non-conservative BoNT/A NTNH variant, a non-conservative Clostridium botulinum serotype B NTNH variant, a non-conservative Clostridium botulinum serotype C1 NTNH variant, a non-conservative Clostridium botulinum serotype D NTNH variant, a non-conservative Clostridium botulinum serotype E NTNH variant, a non-conservative Clostridium botulinum serotype F NTNH variant and a non-conservative Clostridium botulinum serotype G NTNH variant.

As used herein, the term “Clostridial NAP chimeric” means a polypeptide comprising at least a portion of a Clostridial NAP and at least a portion of at least one other polypeptide to form an enhanced targeting domain with at least one property different from the reference Clostridial NAP (see Tables 3-6), with the proviso that this Clostridial NAP chimeric can specifically bind to a Clostridial toxin receptor present in a Clostridial toxin target cell, and thus participate in executing the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate.

As used herein, the term “active Clostridial NAP fragment” means any of a variety of Clostridial NAP fragments comprising the enhanced targeting domain can be useful in aspects of the present invention with the proviso that these NAP fragments can specifically bind to a Clostridial toxin receptor present in a Clostridial toxin target cell, and thus participate in executing the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a binding domain comprises a NAP. In aspects of this embodiment, a NAP comprises a β-trefoil domain derived from a NAP.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a Clostridial HA-33. In an aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a Clostridial HA-33 comprises, e.g., a β-trefoil domain derived from a BoNT/A HA-33, a β-trefoil domain derived from a BoNT/B HA-33, a β-trefoil domain derived from a BoNT/C1 HA-33 or a β-trefoil domain derived from a BoNT/D HA-33. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/B HA-33. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/D HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/D HA-33.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-33 of SEQ ID NO: 11. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 11. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 11. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at least 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at least 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at most 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at most 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at most 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11, at most 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In another embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 11. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at least 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at least 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at least 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at least 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 or at least 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at most 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at most 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at most 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11, at most 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 or at most 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-33 of SEQ ID NO: 12. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 12. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 12. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at least 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at least 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at most 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at most 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at most 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12, at most 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In another embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 12. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at least 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at least 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at least 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at least 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 or at least 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at most 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at most 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at most 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12, at most 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 or at most 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-33 of SEQ ID NO: 13. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 13. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 13. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at least 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at least 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at most 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at most 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at most 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13, at most 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In another embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 13. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at least 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at least 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at least 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at least 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 or at least 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at most 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at most 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at most 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13, at most 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 or at most 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-33 of SEQ ID NO: 14. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-146 or amino acids 153-294 of SEQ ID NO: 14. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 14. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at least 75% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at least 90% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14 or at least 95% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at most 75% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at most 80% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at most 85% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14, at most 90% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14 or at most 95% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In another embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 14. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at least 75% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at least 80% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at least 85% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at least 90% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 or at least 95% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at most 75% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at most 80% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at most 85% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14, at most 90% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 or at most 95% amino acid identity with amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-33 of SEQ ID NO: 15. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-144 or amino acids 151-279 of SEQ ID NO: 15. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 15. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-33 comprises amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at least 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at least 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at most 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at most 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at most 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15, at most 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In another embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 15. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at least 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at least 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at least 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at least 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 or at least 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at most 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at most 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at most 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15, at most 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 or at most 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/B HA-33 of SEQ ID NO: 16. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises amino acids 10-144 or amino acids 151-292 of SEQ ID NO: 16. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 16. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at least 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at least 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at most 75% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at most 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at most 85% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16, at most 90% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In another embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 16. In another aspect of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at least 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at least 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at least 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at least 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 or at least 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at most 75% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at most 80% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at most 85% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16, at most 90% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 or at most 95% amino acid identity with amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/B HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/B HA-33 of SEQ ID NO: 17. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises amino acids 10-146 or amino acids 153-291 of SEQ ID NO: 17. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 17. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-33 comprises amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at least 75% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at least 90% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17 or at least 95% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at most 75% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at most 80% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at most 85% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17, at most 90% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17 or at most 95% amino acid identity with amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In another embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 17. In another aspect of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at least 75% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at least 80% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at least 85% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at least 90% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 or at least 95% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at most 75% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at most 80% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at most 85% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17, at most 90% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 or at most 95% amino acid identity with amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/B HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/C1 HA-33 of SEQ ID NO: 18. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids 10-141 or amino acids 148-285 of SEQ ID NO: 18. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 18. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at least 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at least 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at most 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at most 80% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at most 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18, at most 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In another embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 18. In another aspect of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at least 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at least 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at least 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at least 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 or at least 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 or at most 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/C1 HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/C1 HA-33 of SEQ ID NO: 19. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids 10-141 or amino acids 148-286 of SEQ ID NO: 19. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 19. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at least 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at least 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at most 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at most 80% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at most 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19, at most 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In another embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 19. In another aspect of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at least 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at least 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at least 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at least 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 or at least 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 or at most 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/C1 HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/D HA-33 of SEQ ID NO: 20. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D HA-33 comprises amino acids 10-141 or amino acids 148-286 of SEQ ID NO: 20. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D HA-33 comprises a 1α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/D HA-33 of SEQ ID NO: 20. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D HA-33 comprises amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at least 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at least 80% amino acid identity with amino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at least 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20 or at least 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at most 75% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at most 80% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at most 85% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20, at most 90% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20 or at most 95% amino acid identity with amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In another embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domain of a BoNT/D HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/D HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D HA-33 of SEQ ID NO: 20. In another aspect of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at least 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at least 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at least 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at least 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 or at least 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most 75% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most 80% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most 85% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most 90% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 or at most 95% amino acid identity with amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/D HA-33 comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a Clostridial HA-17. In an aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-17 comprises, e.g., a β-trefoil domain derived from a BoNT/A HA-17, a β-trefoil domain derived from a BoNT/B HA-17, a β-trefoil domain derived from a BoNT/C1 HA-17 or a β-trefoil domain derived from a BoNT/D HA-17. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-17 comprises an α-fold motif of a β-trefoil domain of a BoNT/A HA-17, a β-fold motif of a β-trefoil domain of a BoNT/A HA-17, or a γ-fold motif of a β-trefoil domain of a BoNT/A HA-17. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-17 comprises an α-fold motif of a β-trefoil domain of a BoNT/B HA-17, a β-fold motif of a β-trefoil domain of a BoNT/B HA-17, or a γ-fold motif of a β-trefoil domain of a BoNT/B HA-17. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-17 comprises an α-fold motif of a β-trefoil domain of a BoNT/C1 HA-17, a β-fold motif of a β-trefoil domain of a BoNT/C1 HA-17, or a γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-17. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial HA-17 comprises an α-fold motif of a β-trefoil domain of a BoNT/D HA-17, a β-fold motif of a β-trefoil domain of a BoNT/D HA-17, or a γ-fold motif of a β-trefoil domain of a BoNT/D HA-17.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A HA-17 of SEQ ID NO: 21. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-17 comprises amino acids 9-146 of SEQ ID NO: 21. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A HA-17 comprises a α-fold motif of a β-trefoil domain of a BoNT/A HA-17, a β-fold motif of a β-trefoil domain of a BoNT/A HA-17 or a γ-fold motif of a β-trefoil domain of a BoNT/A HA-17 of SEQ ID NO: 21. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/A HA-17 comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21 or at least 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21 or at most 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.

In another embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-17 of SEQ ID NO: 21. In another aspect of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 or at least 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 or at most 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A HA-17 comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In still other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/B HA-17 of SEQ ID NO: 22. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-17 comprises amino acids 9-146 of SEQ ID NO: 22. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B HA-17 comprises a α-fold motif of a β-trefoil domain of a BoNT/B HA-17, a β-fold motif of a β-trefoil domain of a BoNT/B HA-17 or a γ-fold motif of a β-trefoil domain of a BoNT/B HA-17 of SEQ ID NO: 22. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/B HA-17 comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22 or at least 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22 or at most 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.

In another embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-17 of SEQ ID NO: 22. In another aspect of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 or at least 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 or at most 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/B HA-17 comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In still other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/C1 HA-17 of SEQ ID NO: 23. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-17 comprises amino acids 9-146 of SEQ ID NO: 23. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 HA-17 comprises a α-fold motif of a β-trefoil domain of a BoNT/C1 HA-17, a β-fold motif of a β-trefoil domain of a BoNT/C1 HA-17 or a γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-17 of SEQ ID NO: 23. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/C1 HA-17 comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at least 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at least 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at least 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at least 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23 or at least 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at most 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at most 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at most 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at most 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23 or at most 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23.

In another embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-17 of SEQ ID NO: 23. In another aspect of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at least 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at least 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at least 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at least 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 or at least 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at most 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at most 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at most 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at most 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 or at most 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/C1 HA-17 comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/D HA-17 of SEQ ID NO: 24. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D HA-17 comprises amino acids 9-146 of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D HA-17 comprises a α-fold motif of a β-trefoil domain of a BoNT/D HA-17, a β-fold motif of a β-trefoil domain of a BoNT/D HA-17 or a γ-fold motif of a β-trefoil domain of a BoNT/D HA-17 of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/D HA-17 comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at least 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at least 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at least 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at least 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24 or at least 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at most 75% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at most 80% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at most 85% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at most 90% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24 or at most 95% amino acid identity with amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24.

In another embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/D HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D HA-17 of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at least 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at least 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at least 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at least 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 or at least 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at most 75% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at most 80% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at most 85% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at most 90% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 or at most 95% amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/D HA-17 comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In still other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a Clostridial NTNH. In an aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a Clostridial NTNH comprises, e.g., a β-trefoil domain derived from a BoNT/A NTNH, a β-trefoil domain derived from a BoNT/B NTNH, a β-trefoil domain derived from a BoNT/C1 NTNH, a β-trefoil domain derived from a BoNT/D NTNH, a β-trefoil domain derived from a BoNT/E NTNH, a β-trefoil domain derived from a BoNT/F NTNH, or a β-trefoil domain derived from a BoNT/G NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an α-fold motif of a β-trefoil domain of a BoNT/A NTNH, β-fold motif of β-trefoil domain of a BoNT/A NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/A NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an α-fold motif of a β-trefoil domain of a BoNT/B NTNH, a β-fold motif of a β-trefoil domain of a BoNT/B NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/B NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an α-fold motif of a β-trefoil domain of a BoNT/C1 NTNH, a β-fold motif of a β-trefoil domain of a BoNT/C1 NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/C1 NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an β-fold motif of a β-trefoil domain of a BoNT/D NTNH, a β-fold motif of a β-trefoil domain of a BoNT/D NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/D NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an β-fold motif of a β-trefoil domain of a BoNT/E NTNH, a β-fold motif of a β-trefoil domain of a BoNT/E NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/E NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an α-fold motif of a β-trefoil domain of a BoNT/F NTNH, a β-fold motif of a β-trefoil domain of a BoNT/F NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/F NTNH. In another aspect of this embodiment, a β-trefoil domain derived from a Clostridial NTNH comprises an α-fold motif of a β-trefoil domain of a BoNT/G NTNH, a β-fold motif of a β-trefoil domain of a BoNT/G NTNH, or a γ-fold motif of a β-trefoil domain of a BoNT/G NTNH.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A NTNH of SEQ ID NO: 25. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1194 of SEQ ID NO: 25. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25 or at least 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at most 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at most 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at most 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at most 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25 or at most 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25.

In another embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 75% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 80% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 85% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 90% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 or at least 95% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 75% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 80% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 85% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 90% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 or at most 95% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A NTNH of SEQ ID NO: 26. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1199 of SEQ ID NO: 26. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at least 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at least 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at least 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at least 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26 or at least 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at most 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at most 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at most 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at most 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26 or at most 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26.

In another embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 75% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 80% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 85% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 90% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 or at least 95% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at most 75% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at most 80% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at most 85% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at most 90% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 or at most 95% amino acid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/A NTNH of SEQ ID NO: 27. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1194 of SEQ ID NO: 27. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at least 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at least 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at least 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at least 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27 or at least 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at most 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at most 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at most 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at most 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27 or at most 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27.

In another embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 75% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 80% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 85% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 90% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 or at least 95% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at most 75% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at most 80% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at most 85% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at most 90% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 or at most 95% amino acid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/A NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In still other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In other aspects of this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/B NTNH of SEQ ID NO: 28. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B NTNH comprises amino acids 1049-1198 of SEQ ID NO: 28. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/B NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/B NTNH, a β-fold motif of a β-trefoil domain of a BoNT/B NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/B NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/B NTNH comprises amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at least 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at least 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at least 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at least 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28 or at least 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at most 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at most 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at most 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at most 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28 or at most 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In still other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In still other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28.

In another embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/B NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 or at least 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 or at most 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In still other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/B NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In still other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects of this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/C1 NTNH of SEQ ID NO: 29. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 NTNH comprises amino acids 1049-1197 of SEQ ID NO: 29. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/C1 NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/C1 NTNH, a β-fold motif of a β-trefoil domain of a BoNT/C1 NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/C1 NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/C1 NTNH comprises amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at least 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at least 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at least 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at least 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29 or at least 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at most 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at most 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at most 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at most 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29 or at most 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29.

In another embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1 NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 or at least 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at most 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at most 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at most 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at most 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 or at most 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/C1 NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In still other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/D NTNH of SEQ ID NO: 30. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D NTNH comprises amino acids 1049-1197 of SEQ ID NO: 30. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/D NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/D NTNH, a β-fold motif of a β-trefoil domain of a BoNT/D NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/D NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/D NTNH comprises amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at least 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at least 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at least 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at least 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30 or at least 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at most 75% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at most 80% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at most 85% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at most 90% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30 or at most 95% amino acid identity with amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In still other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In still other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30.

In another embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/D NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 or at least 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at most 75% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at most 80% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at most 85% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at most 90% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 or at most 95% amino acid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In still other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/D NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In still other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In other aspects of this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/E NTNH of SEQ ID NO: 31. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/E NTNH comprises amino acids 1014-1163 of SEQ ID NO: 31. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/E NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/E NTNH, a β-fold motif of a β-trefoil domain of a BoNT/E NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/E NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/E NTNH comprises amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at least 75% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at least 80% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at least 85% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at least 90% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31 or at least 95% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In yet other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at most 75% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at most 80% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at most 85% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at most 90% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31 or at most 95% amino acid identity with amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In yet other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In still other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In yet other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In still other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31.

In another embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a β4/β5 hairpin turn of β-trefoil domain of a BoNT/E NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/E NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 75% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 80% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 85% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 90% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 or at least 95% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In yet other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at most 75% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at most 80% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at most 85% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at most 90% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 or at most 95% amino acid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In still other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/E NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In still other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In other aspects of this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/F NTNH of SEQ ID NO: 32. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/F NTNH comprises amino acids 1016-1160 of SEQ ID NO: 32. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/F NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/F NTNH, a β-fold motif of a β-trefoil domain of a BoNT/F NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/F NTNH comprises amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at least 75% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at least 80% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at least 85% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at least 90% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32 or at least 95% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at most 75% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at most 80% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at most 85% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at most 90% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32 or at most 95% amino acid identity with amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32.

In another embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/F NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 75% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 80% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 85% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 90% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 or at least 95% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at most 75% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at most 80% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at most 85% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at most 90% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 or at most 95% amino acid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/F NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In another embodiment, a binding domain comprises a β-trefoil domain derived from a BoNT/F NTNH of SEQ ID NO: 33. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/F NTNH comprises amino acids 1017-1166 of SEQ ID NO: 33. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/F NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/F NTNH, a β-fold motif of a β-trefoil domain of a BoNT/F NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/F NTNH comprises amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at least 75% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at least 80% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at least 85% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at least 90% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33 or at least 95% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at most 75% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at most 80% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at most 85% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at most 90% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33 or at most 95% amino acid identity with amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33.

In another embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/F NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 75% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 80% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 85% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 90% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 or at least 95% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at most 75% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at most 80% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at most 85% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at most 90% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 or at most 95% amino acid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/F NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In still other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In other aspects of this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/G NTNH of SEQ ID NO: 34. In another embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/G NTNH comprises amino acids 1050-1199 of SEQ ID NO: 34. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a BoNT/G NTNH comprises a α-fold motif of a β-trefoil domain of a BoNT/G NTNH, a β-fold motif of a β-trefoil domain of a BoNT/G NTNH or a γ-fold motif of a β-trefoil domain of a BoNT/G NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domain derived from a BoNT/G NTNH comprises amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at least 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at least 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at least 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at least 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34 or at least 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In yet other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at most 75% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at most 80% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at most 85% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at most 90% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34 or at most 95% amino acid identity with amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In yet other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In still other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In yet other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In still other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34.

In another embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/G NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/G NTNH of SEQ ID NO: 24. In another aspect of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 75% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 80% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 85% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 90% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 or at least 95% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In yet other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at most 75% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at most 80% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at most 85% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at most 90% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 or at most 95% amino acid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In still other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 can be replaced with phenylalanine. In yet other aspects of this embodiment, a β-trefoil domain with enhanced binding activity derived from a BoNT/G NTNH comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In still other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In other aspects of this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

Another example of a binding domain, includes, without limitation, ligands that bind the same receptors as does the naturally-occurring Clostridial toxins. Recent studies are revealing components of the endogenous receptors used by a Clostridial toxin during the intoxication process. As a non-limiting example, fibroblast growth factor 3 receptor (FGFR3) serves as a BoNT/A receptor, see, e.g., Ester Fernandez-Salas et al., Botulinum Toxin Screening Assays, PCT Patent Application No. 2005/006421 (Sep. 9, 2005). As another non-limiting example, synaptotagmin I serves as a BoNT/B receptor and as a BoNT/G receptor, see, e.g., Min Dong et al., Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells, 162(7) J. Cell Biol. 1293-1303 (2003); and Andreas Rummel et al., Synaptotagmins I and II act as nerve cell receptors for botulinum neurotoxin G, 279(29) J. Biol. Chem. 30865-30870 (2004). As yet another non-limiting example, synaptotagmin II serves as a BoNT/B receptor and as a BoNT/G receptor, see, e.g., Min Dong et al., supra, (2003); and Andreas Rummel et al., supra, (2004). The selection of a binding domain will depend on the Clostridial toxin being modified. As non-limiting examples, ligands that selectively bind to a FGFR3 could be used as a binding domain for a modified BoNT/A, whereas ligands that selectively bind a Synaptotagmin I or Synaptotagmin II could be used as a binding domain for a modified BoNT/B or a modified BoNT/G.

In addition to its enhanced targeting activity, replacement of a naturally-occurring binding domain with, e.g., an FGF ligand, has an added advantage of reducing the likelihood of the modified toxin from eliciting an immunogenic response. Regions found in the H_(CC) targeting domain are bound by neutralizing anti-BoNT/A antibodies, see, e.g., M. Zouhair Atassi et al., Mapping of the Antibody-binding Regions on Botulinum Neurotoxin H-chain Domain 855-1296 with Anti-toxin Antibodies from Three Host Species, 15 J. PROT. CHEM. 691-700, (1996); M. Zouhair Atassi & Behzod Z. Dolimbek, Mapping of the Antibody-binding Profile on Botulinum Neurotoxin A H _(N)-domain (residues 449-859) with Anti-toxin Antibodies from Four Host Species. Antigenic Profile of the Entire H-chain of Botulinum Neurotoxin A, 23(1) PROTEIN J. 39-52, (2004). Therefore, elimination of this binding domain will reduce the likelihood of an immunogenic response because 1) the Clostridial toxin H_(CC) binding domain is absent; 2) a human FGF binding domain will most likely not elicit an immunogenic response in a patient because it is a human polypeptide.

Fibroblast growth factors (FGF) participate in many developmental, differentiation and growth and repair processes of cells through complex combinatorial signaling pathways. Presently, at least 23 ligands (FGF1-23) are known to signal through a family of five transmembrane tyrosine kinase FGF receptors (FGFR1-5). Affinity of FGFRs for their ligands is highly diverse with different affinities for each family member of growth factors, see, e.g., C. J. Powers et al., Fibroblast growth factors, their receptors and signaling, 7(3) Endocr. Relat. Cancer. 165-197 (2000). This diversity is achieved in part by the generation of alternatively spliced variants encoding distinct receptor isoforms, see, e.g., Bernhard Reuss & Oliver von Bohlen und Halbach, Fibroblast growth factors and their receptors in the central nervous system, 313(2) Cell Tissue Res. 139-157 (2003). The protein region that appears to have the highest influence on ligand binding selectivity is a portion of the IgIII domain, for which isoforms encoded by three different splice variants have been identified. These three isoforms, designated IgIIIa, IgIIIb and IgIIIc, have relative binding affinities for different FGFR family members. Alternative splicing in the FGFR ligand binding domain, designated a and b, generates additional receptor isoforms with novel ligand affinities. Isoforms for IgIIIa, IgIIIb and IgIIIc have been identified for both FGFR1 and FGFR2. Thus far, the IgIIIa isoform of FGFR3 and the IgIIIa and IgIIIb isoforms of FGFR4 and FGFR5 have not been reported.

Currently, several FGFs have been shown to selectively bind FGFR3, such as, e.g., FGF-1, FGF-2, FGF-4, FGF-8, FGF-9, FGF-17 and FGF-18, see, e.g., Hecht et al., 1995; Ornitz et al., 1996; and Xu et al., 2000. Additional studies have revealed that FGF-1 and FGF9 preferentially bind FGFR3IIIb, whereas FGF-1 FGF-2, FGF-4, FGF-8, and FGF9 preferentially bind FGFR3IIIc. Studies have shown that each of these ligands is present in various vertebrate species with a high degree of sequence identity which suggests functional equivalence or similarity. As a non-limiting example, FGF-8 is found in fish, birds and mammals and each of these FGF-8 ligands have over 80% amino acid sequence identity. As another non-limiting example, FGF-18 is found in fish, birds and mammals and each of these FGF-18 ligands have over 70% amino acid sequence identity. Crystallographic studies have revealed that all FGFs are structurally organized into β-trefoil domain. The amino acid sequences comprising the β-trefoil domains found in various FGF ligands that bind FGFR3 are shown in Table 7.

TABLE 7 β-trefoil Domains of FGFs Amino Acid Sequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Ligand SEQ ID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold FGF-1 35 26-64 65-67 68-105 106-108 109-155 FGF-2 36 29-67 68-70 71-111 112-114 115-155 FGF-4 37  83-121 122-124 125-162  163-165 166-206 FGF-8 38 43-80 81-83 84-123 124-126 127-172 FGF-9 39  63-100 101-103 104-144  145-147 148-196 FGF-17 40 55-91 92-94 95-134 135-137 138-183 FGF-18 41 54-91 92-94 95-134 135-137 138-183

As used herein, the term “Fibroblast Growth Factor” is synonymous with “FGF” and means a polypeptide with selective binding activity, such as, e.g., a binding affinity or a binding specificity, for a receptor, including endogenous Clostridial toxin receptors such as, e.g., a receptor comprising FGFR3. It is envisioned that both naturally occurring FGFs as well as non-naturally occurring FGFs can be used as a binding domain. Any of a variety of sequence alignment methods can be used to determine percent identity of a non-naturally occurring FGF relative to a naturally-occurring FGF, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.

Approaches well known to one skilled in the art on how to modify a FGF in order to increase its binding activity for an endogenous Clostridial toxin receptor present on a naturally-occurring Clostridial toxin target cell. As described above, one approach involves identifying amino acids using computational protein design algorithms; changing specifically-identified amino acids using, without limitation, site-directed mutagenesis, oligonucleotide-directed mutagenesis and site-specific mutagenesis; and testing the binding activity of multivalent Clostridial toxins comprising a modified FGF with enhanced binding activity using, e.g., heterogeneous assays, homogeneous assays and non-separating homogeneous assays. It is further envisioned that the binding activity of a multivalent Clostridial toxin disclosed in the present specification can be determined by affinity chromatography using immobilized receptors and interfacial optical assays. In another approach described above, a binding activity of a modified FGF for a naturally-occurring Clostridial toxin receptor present on a naturally-occurring Clostridial toxin target cell can be achieved using directed-evolution methods.

A FGF includes, without limitation, naturally occurring FGF variants, such as, e.g., FGF isoforms and FGF subtypes; non-naturally occurring FGF variants, such as, e.g., conservative FGF variants, non-conservative FGF variants, FGF chimerics, active FGF fragments thereof, or any combination thereof.

As used herein, the term “FGF variant,” whether naturally-occurring or non-naturally-occurring, means a FGF that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Table 7) and can be described in percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all FGF variants disclosed in the present specification are capable of executing the cell binding step of the intoxication process.

It is recognized by those of skill in the art that there can be naturally occurring FGF variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently at least four FGF-8 variants, FGF-8A, FGF-8A, FGF-8B, FGF-8E and FGF-8F, with specific FGF-8 variants showing various degrees of amino acid divergence when compared to another FGF-8 variant. As another example, there are presently at least two FGF-2 variants, FGF-2-1 and FGF-2-1, with the FGF-2-1 variant showing an amino acid divergence when compared to the FGF-2-2 variant. As used herein, the term “naturally occurring FGF variant” means any FGF produced by a naturally-occurring process, including, without limitation, FGF isoforms produced from alternatively-spliced transcripts, FGF isoforms produced by spontaneous mutation and FGF subtypes. A naturally occurring FGF variant can function in substantially the same manner as the reference FGF on which the naturally occurring FGF variant is based, and can be substituted for the reference FGF in any aspect of the present invention. A naturally occurring FGF variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids from the reference FGF on which the naturally occurring FGF variant is based. A naturally occurring FGF variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference FGF on which the naturally occurring FGF variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference FGF on which the naturally occurring FGF variant is based.

A non-limiting examples of a naturally occurring FGF variant is a FGF isoform such as, e.g., a FGF-1 isoform, a FGF-2 isoform, a FGF-4 isoform, a FGF-8 isoform, a FGF-9 isoform, a FGF-17 isoform and a FGF-18 isoform. A FGF isoform can function in substantially the same manner as the reference FGF on which the FGF isoform is based, and can be substituted for the reference FGF in any aspect of the present invention.

Another non-limiting examples of a naturally occurring FGF variant is a FGF subtype such as, e.g., a FGF-1 subtype, a FGF-2 subtype, a FGF-4 subtype, a FGF-8 subtype, a FGF-9 subtype, a FGF-17 subtype and a FGF-18 subtype. A FGF subtype can function in substantially the same manner as the reference FGF on which the FGF subtype is based, and can be substituted for the reference FGF in any aspect of the present invention.

As used herein, the term “non-naturally occurring FGF variant” means any FGF produced with the aid of human manipulation, including, without limitation, FGFs produced by genetic engineering using random mutagenesis or rational design and FGFs produced by chemical synthesis. Non-limiting examples of non-naturally occurring FGF variants include, e.g., conservative FGF variants, non-conservative FGF variants, FGF chimeric variants and active FGF fragments.

As used herein, the term “conservative FGF variant” means a FGF that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference FGF sequence (see Table 7). Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative FGF variant can function in substantially the same manner as the reference FGF on which the conservative FGF variant is based, and can be substituted for the reference FGF in any aspect of the present invention. A conservative FGF variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids from the reference FGF on which the conservative FGF variant is based. A conservative FGF variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference FGF on which the conservative FGF variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference FGF on which the conservative FGF variant is based. Non-limiting examples of a conservative FGF variant include, e.g., a conservative FGF-1 variant, a conservative FGF-2 variant, a conservative FGF-4 variant, a conservative FGF-8 variant, a conservative FGF-9 variant, a conservative FGF-17 variant and a conservative FGF-18 variant.

As used herein, the term “non-conservative FGF variant” means a FGF in which 1) at least one amino acid is deleted from the reference FGF on which the non-conservative FGF variant is based; 2) at least one amino acid added to the reference FGF on which the non-conservative FGF is based; or 3) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to that of the original amino acid from the reference FGF sequence (see Table 7). A non-conservative FGF variant can function in substantially the same manner as the reference FGF on which the non-conservative FGF variant is based, and can be substituted for the reference FGF in any aspect of the present invention. A non-conservative FGF variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference FGF on which the non-conservative FGF variant is based. A non-conservative FGF variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference FGF on which the non-conservative FGF variant is based. A non-conservative FGF variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids from the reference FGF on which the non-conservative FGF variant is based. A non-conservative FGF variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference FGF on which the non-conservative FGF variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference FGF on which the non-conservative FGF variant is based. Non-limiting examples of a non-conservative FGF variant include, e.g., a non-conservative FGF-1 variant, a non-conservative FGF-2 variant, a non-conservative FGF-4 variant, a non-conservative FGF-8 variant, a non-conservative FGF-9 variant, a non-conservative FGF-17 variant and a non-conservative FGF-18 variant.

As used herein, the term “FGF chimeric” means a polypeptide comprising at least a portion of a FGF and at least a portion of at least one other polypeptide to form an enhanced targeting domain with at least one property different from the reference FGF (see Table 7), with the proviso that this FGF chimeric can specifically bind to an endogenous Clostridial toxin receptor present in a Clostridial toxin target cell, and thus participate in executing the overall cellular mechanism whereby a Clostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a binding domain comprises a FGF. In aspects of this embodiment, a FGF comprises a β-trefoil domain derived from a FGF. In an aspect of this embodiment, a β-trefoil domain derived from a FGF comprises, e.g., a β-trefoil domain derived from a FGF-1, a β-trefoil domain derived from a FGF-2, a β-trefoil domain derived from FGF-4, a β-trefoil domain derived from a FGF-8, a β-trefoil domain derived from a FGF-9, a β-trefoil domain derived from a FGF-17 or a β-trefoil domain derived from a FGF-18. In another aspect of this embodiment, a β-trefoil domain derived from a FGF comprises a α-fold motif of a β-trefoil domain of a FGF, a β-fold motif of a β-trefoil domain of a FGF or a γ-fold motif of a β-trefoil domain of a FGF.

In another embodiment, a binding domain comprises a β-trefoil domain derived a FGF-1 of SEQ ID NO: 35. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-1 comprises amino acids 26-155 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-1 comprises an α-fold motif of a β-trefoil domain of a FGF-1, a β-fold motif of a β-trefoil domain of a FGF-1 or a γ-fold motif of a β-trefoil domain of a FGF-1 of SEQ ID NO: 35. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-1 comprises amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at least 75% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at least 80% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at least 85% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at least 90% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35 or at least 95% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at most 75% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at most 80% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at most 85% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at most 90% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35 or at most 95% amino acid identity with amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In still other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In still other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35.

In another embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-1 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-1 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at least 75% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at least 80% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at least 85% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at least 90% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 or at least 95% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at most 75% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at most 80% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at most 85% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at most 90% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 or at most 95% amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In still other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In still other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a binding domain comprising a FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In another embodiment, a binding domain comprises a β-trefoil domain derived a FGF-2 of SEQ ID NO: 36. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-2 comprises amino acids 29-155 of SEQ ID NO: 36. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-2 comprises an α-fold motif of a β-trefoil domain of a FGF-2, a β-fold motif of a β-trefoil domain of a FGF-2 or a γ-fold motif of a β-trefoil domain of a FGF-2 of SEQ ID NO: 36. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-2 comprises amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at least 75% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at least 80% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at least 85% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at least 90% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36 or at least 95% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at most 75% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at most 80% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at most 85% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at most 90% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36 or at most 95% amino acid identity with amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In still other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In still other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36.

In another embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-2 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-2 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at least 75% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at least 80% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at least 85% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at least 90% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 or at least 95% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at most 75% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at most 80% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at most 85% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at most 90% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 or at most 95% amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In still other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In still other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a binding domain comprising a FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In another embodiment, a binding domain comprises a β-trefoil domain derived a FGF-4 of SEQ ID NO: 37. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-4 comprises amino acids 83-206 of SEQ ID NO: 37. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-4 comprises an α-fold motif of a β-trefoil domain of a FGF-4, a β-fold motif of a β-trefoil domain of a FGF-4 or a γ-fold motif of a β-trefoil domain of a FGF-4 of SEQ ID NO: 37. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-4 comprises amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at least 75% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at least 80% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at least 85% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at least 90% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37 or at least 95% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at most 75% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at most 80% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at most 85% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at most 90% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37 or at most 95% amino acid identity with amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In still other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In still other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37.

In another embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-4 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-4 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at least 75% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at least 80% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at least 85% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at least 90% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 or at least 95% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at most 75% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at most 80% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at most 85% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at most 90% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 or at most 95% amino acid identity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In still other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In still other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a binding domain comprising a FGF-4 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37.

In another embodiment, a binding domain comprises a β-trefoil domain derived a FGF-8 of SEQ ID NO: 38. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-8 comprises amino acids 43-172 of SEQ ID NO: 38. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-8 comprises an α-fold motif of a β-trefoil domain of a FGF-8, a β-fold motif of a β-trefoil domain of a FGF-8 or a γ-fold motif of a β-trefoil domain of a FGF-8 of SEQ ID NO: 38. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-8 comprises amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at least 75% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at least 80% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at least 85% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at least 90% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38 or at least 95% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at most 75% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at most 80% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at most 85% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at most 90% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38 or at most 95% amino acid identity with amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In still other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In still other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38.

In another embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-8 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-8 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at least 75% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at least 80% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at least 85% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at least 90% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 or at least 95% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at most 75% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at most 80% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at most 85% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at most 90% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 or at most 95% amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In still other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In still other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a binding domain comprising a FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In another embodiment, a binding domain comprises a β-trefoil domain derived a FGF-9 of SEQ ID NO: 39. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-9 comprises amino acids 63-196 of SEQ ID NO: 39. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-9 comprises an α-fold motif of a β-trefoil domain of a FGF-9, a β-fold motif of a β-trefoil domain of a FGF-9 or a γ-fold motif of a β-trefoil domain of a FGF-9 of SEQ ID NO: 39. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-9 comprises amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at least 75% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at least 80% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at least 85% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at least 90% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39 or at least 95% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at most 75% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at most 80% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at most 85% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at most 90% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39 or at most 95% amino acid identity with amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In still other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In still other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39.

In another embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-9 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-9 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at least 75% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at least 80% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at least 85% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at least 90% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 or at least 95% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at most 75% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at most 80% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at most 85% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at most 90% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 or at most 95% amino acid identity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In still other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In still other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a binding domain comprising a FGF-9 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39.

In another embodiment, a binding domain comprises β-trefoil domain derived a FGF-17 of SEQ ID NO: 40. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-17 comprises amino acids 55-183 of SEQ ID NO: 40. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-17 comprises an α-fold motif of a β-trefoil domain of a FGF-17, a β-fold motif of a β-trefoil domain of a FGF-17 or a γ-fold motif of a β-trefoil domain of a FGF-17 of SEQ ID NO: 40. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-17 comprises amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at least 75% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at least 80% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at least 85% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at least 90% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40 or at least 95% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at most 75% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at most 80% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at most 85% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at most 90% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40 or at most 95% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In still other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In still other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40.

In another embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-17 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-17 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at least 75% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at least 80% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at least 85% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at least 90% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 or at least 95% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at most 75% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at most 80% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at most 85% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at most 90% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 or at most 95% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In still other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In still other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of this embodiment, a binding domain comprising a FGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40.

In another embodiment, a binding domain comprises β-trefoil domain derived a FGF-18 of SEQ ID NO: 41. In another embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-18 comprises amino acids 54-183 of SEQ ID NO: 41. In another aspect of this embodiment, a binding domain comprising a β-trefoil domain derived from a FGF-18 comprises an α-fold motif of a β-trefoil domain of a FGF-18, a β-fold motif of a β-trefoil domain of a FGF-18 or a γ-fold motif of a β-trefoil domain of a FGF-18 of SEQ ID NO: 41. In another aspect of this embodiment, a β-trefoil domain derived from a FGF-18 comprises amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at least 75% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at least 80% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at least 85% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at least 90% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41 or at least 95% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at most 75% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at most 80% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at most 85% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at most 90% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41 or at most 95% amino acid identity with amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In still other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In still other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41.

In another embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-18 or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-18 of SEQ ID NO: 35. In another aspect of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at least 75% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at least 80% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at least 85% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at least 90% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 or at least 95% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at most 75% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at most 80% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at most 85% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at most 90% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 or at most 95% amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a non-contiguous amino acid substitution of any amino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 can be replaced with glycine. In other aspects of this embodiment, a non-contiguous amino acid substitution of any hydrophobic amino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In still other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, contiguous amino acid substitutions of amino acids from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 can be replaced with glycine. In other aspects of this embodiment, contiguous amino acid substitutions of hydrophobic amino acids from amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 can be replaced with phenylalanine. In yet other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In still other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of this embodiment, a binding domain comprising a FGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41.

Because of the modular nature of the β-trefoil domain, it is envisioned that any combination of α-folds, β-folds, γ-folds, β4/β5 β-hairpin turns, β8/β9 β-hairpin turns, or any combination thereof from a β-trefoil domain of a Clostridial toxin binding domain, a β-trefoil domain of a NAP or a β-trefoil domain, a β-trefoil domain of an FGF ligand, or any combination thereof, can be used to practice aspect of the present invention. As a non-limiting example, a multivalent Clostridial toxin disclosed in the present specification can comprise a binding domain comprising a α-fold from a BoNT/A binding domain, a β-fold from a FGF-18 and a 2γ-fold from a BoNT/A HA-33. As another non-limiting example, a multivalent Clostridial toxin disclosed in the present specification can comprise a binding domain comprising a α-fold from a BoNT/A HA-17, a β-fold from a Clostridium botulinum serotype E NTNH and a γ-fold from a modified BoNT/C1 binding domain with enhanced binding activity.

Thus, in an embodiment, a binding domain comprises a glycogen-like peptide. In another embodiment, a binding domain comprises a glycogen-like peptide of SEQ ID NO: 42. In another embodiment, a binding domain is derived from a glycogen-like peptide. In another embodiment, a binding domain is derived from a glycogen-like peptide of SEQ ID NO: 42. In aspects of this embodiment, a binding domain is derived from a glycogen-like peptide comprises a GRPP, a GLP-1, a GLP-2, a glucagon or an oxyntomodulin. In aspects of this embodiment, a binding domain is derived from a glycogen-like peptide comprising amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at least 75% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at least 80% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at least 85% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at least 90% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42 or at least 95% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In yet other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at most 75% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at most 80% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at most 85% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at most 90% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42 or at most 95% amino acid identity with amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In yet other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In still other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In yet other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In still other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects of this embodiment, a glycogen-like peptide comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In an embodiment, a binding domain comprises a PACAP. In another embodiment, a binding domain comprises a PACAP of SEQ ID NO: 43. In another embodiment, a binding domain is derived from a PACAP. In another embodiment, a binding domain is derived from a PACAP of SEQ ID NO: 43. In an aspect of this embodiment, a binding domain is derived from a PACAP comprising amino acids 132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at least 75% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at least 80% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at least 85% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at least 90% amino acid identity with amino acids 132-158 of SEQ ID NO: 43 or at least 95% amino acid identity with amino acids 132-158 of SEQ ID NO: 43. In yet other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at most 75% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at most 80% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at most 85% amino acid identity with amino acids 132-158 of SEQ ID NO: 43, at most 90% amino acid identity with amino acids 132-158 of SEQ ID NO: 43 or at most 95% amino acid identity with amino acids 132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 132-158 of SEQ ID NO: 43. In yet other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 132-158 of SEQ ID NO: 43. In still other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 132-158 of SEQ ID NO: 43. In yet other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 132-158 of SEQ ID NO: 43. In still other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 132-158 of SEQ ID NO: 43.

In an embodiment, a binding domain comprises a GHRH. In another embodiment, a binding domain comprises a GHRH of SEQ ID NO: 44. In another embodiment, a binding domain is derived from a GHRH. In another embodiment, a binding domain is derived from a GHRH of SEQ ID NO: 44. In aspects of this embodiment, a binding domain is derived from a GHRH comprising amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at least 75% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at least 80% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at least 85% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at least 90% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44 or at least 95% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In yet other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 75% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 80% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 85% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 90% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44 or at most 95% amino acid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In yet other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In still other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In yet other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In still other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44.

In an embodiment, a binding domain comprises a VIP1. In another embodiment, a binding domain comprises a VIP1 of SEQ ID NO: 45. In another embodiment, a binding domain is derived from a VIP1. In another embodiment, a binding domain is derived from a VIP1 of SEQ ID NO: 45. In aspects of this embodiment, a binding domain is derived from a VIP1 comprising amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at least 75% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at least 80% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at least 85% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at least 90% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45 or at least 95% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In yet other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at most 75% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at most 80% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at most 85% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at most 90% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45 or at most 95% amino acid identity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In yet other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In still other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In yet other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In still other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In an embodiment, a binding domain comprises a VIP2. In another embodiment, a binding domain comprises a VIP2 of SEQ ID NO: 46. In another embodiment, a binding domain is derived from a VIP2. In another embodiment, a binding domain is derived from a VIP2 of SEQ ID NO: 46. In aspects of this embodiment, a binding domain is derived from a VIP2 comprising amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at least 75% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at least 80% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at least 85% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at least 90% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46 or at least 95% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In yet other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at most 75% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at most 80% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at most 85% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at most 90% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46 or at most 95% amino acid identity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In yet other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In still other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In yet other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In still other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In an embodiment, a binding domain comprises a GIP. In another embodiment, a binding domain comprises a GIP of SEQ ID NO: 47. In another embodiment, a binding domain is derived from a GIP. In another embodiment, a binding domain is derived from a GIP of SEQ ID NO: 47. In aspects of this embodiment, a binding domain is derived from a GIP comprising amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at least 75% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at least 80% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at least 85% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at least 90% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47 or at least 95% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In yet other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 75% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 80% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 85% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 90% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47 or at most 95% amino acid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In yet other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In still other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In yet other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In still other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In an embodiment, a binding domain comprises a Secretin. In another embodiment, a binding domain comprises a Secretin of SEQ ID NO: 48. In another embodiment, a binding domain is derived from a Secretin. In another embodiment, a binding domain is derived from a Secretin of SEQ ID NO: 48. In an aspect of this embodiment, a binding domain is derived from a Secretin comprising amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at least 75% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at least 80% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at least 85% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at least 90% amino acid identity with amino acids 28-54 of SEQ ID NO: 48 or at least 95% amino acid identity with amino acids 28-54 of SEQ ID NO: 48. In yet other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 75% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 80% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 85% amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 90% amino acid identity with amino acids 28-54 of SEQ ID NO: 48 or at most 95% amino acid identity with amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 28-54 of SEQ ID NO: 48. In yet other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 28-54 of SEQ ID NO: 48. In still other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 28-54 of SEQ ID NO: 48. In yet other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 28-54 of SEQ ID NO: 48. In still other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 28-54 of SEQ ID NO: 48.

In an embodiment, a binding domain comprises a Gastrin. In another embodiment, a binding domain comprises a Gastrin of SEQ ID NO: 49. In another embodiment, a binding domain is derived from a Gastrin. In another embodiment, a binding domain is derived from a Gastrin of SEQ ID NO: 49. In aspects of this embodiment, a binding domain is derived from a Gastrin comprising amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at least 75% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at least 80% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at least 85% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at least 90% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49 or at least 95% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In yet other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most 75% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most 80% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most 85% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most 90% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49 or at most 95% amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In yet other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In still other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In yet other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In still other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49.

In an embodiment, a binding domain comprises a GRP. In another embodiment, a binding domain comprises a GRP of SEQ ID NO: 50. In another embodiment, a binding domain is derived from a GRP. In another embodiment, a binding domain is derived from a GRP of SEQ ID NO: 50. In aspects of this embodiment, a binding domain is derived from a GRP comprising amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at least 75% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at least 80% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at least 85% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at least 90% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50 or at least 95% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In yet other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 75% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 80% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 85% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 90% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50 or at most 95% amino acid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In yet other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In still other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In yet other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In still other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In an embodiment, a binding domain comprises a CCK. In another embodiment, a binding domain comprises a CCK of SEQ ID NO: 51. In another embodiment, a binding domain is derived from a CCK. In another embodiment, a binding domain is derived from a CCK of SEQ ID NO: 51. In an aspect of this embodiment, a binding domain is derived from a CCK comprising amino acids 99-112 of SEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at least 75% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at least 80% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at least 85% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at least 90% amino acid identity with amino acids 99-112 of SEQ ID NO: 51 or at least 95% amino acid identity with amino acids 99-112 of SEQ ID NO: 51. In yet other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at most 75% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at most 80% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at most 85% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, at most 90% amino acid identity with amino acids 99-112 of SEQ ID NO: 51 or at most 95% amino acid identity with amino acids 99-112 of SEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 99-112 of SEQ ID NO: 51. In yet other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 99-112 of SEQ ID NO: 51. In still other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 99-112 of SEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 99-112 of SEQ ID NO: 51. In yet other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 99-112 of SEQ ID NO: 51. In still other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 99-112 of SEQ ID NO: 51.

Another example of a binding domain disclosed in the present specification is, e.g., neurohormones, such as, e.g., corticotropin-releasing hormone (CCRH) or parathyroid hormone (PTH).

Thus, in an embodiment, a binding domain comprises a CCRH. In another embodiment, a binding domain comprises a CCRH of SEQ ID NO: 52. In another embodiment, a binding domain is derived from a CCRH. In another embodiment, a binding domain is derived from a CCRH of SEQ ID NO: 52. In aspects of this embodiment, a binding domain is derived from a CCRH comprising amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at least 75% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at least 80% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at least 85% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at least 90% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52 or at least 95% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In yet other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at most 75% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at most 80% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at most 85% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at most 90% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52 or at most 95% amino acid identity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In yet other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In still other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In yet other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In still other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In an embodiment, a binding domain comprises a PTH. In another embodiment, a binding domain comprises a PTH of SEQ ID NO: 53. In another embodiment, a binding domain is derived from a PTH. In another embodiment, a binding domain is derived from a PTH of SEQ ID NO: 53. In aspects of this embodiment, a binding domain is derived from a PTH comprising amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at least 75% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at least 80% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at least 85% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at least 90% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53 or at least 95% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In yet other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at most 75% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at most 80% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at most 85% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at most 90% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53 or at most 95% amino acid identity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In yet other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In still other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In yet other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In still other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTH comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

Another example of a binding domain disclosed in the present specification is, e.g., neuroregulatory cytokines, such as, e.g., ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemia inhibitory factor (LIF), also known as cholinergic differentiation factor (CDF), interleukins (ILs), like IL1, IL2, IL6, IL8 and IL10, onostatin M, cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), or neuroleukin, also known as glucose phosphate isomerase (GPI), autocrine motility factor (AMF), maturation and differentiation factor (MF).

Thus, in an embodiment, a binding domain comprises a CNTF. In another embodiment, a binding domain comprises a CNTF of SEQ ID NO: 54. In another embodiment, a binding domain is derived from a CNTF. In another embodiment, a binding domain is derived from a CNTF of SEQ ID NO: 54. In aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 54 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 54. In yet other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 54, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 54 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 54.

In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 54. In yet other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 54. In still other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 54.

In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 54. In yet other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 54. In still other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 54.

In an embodiment, a binding domain comprises a GPA. In another embodiment, a binding domain comprises a GPA of SEQ ID NO: 55. In another embodiment, a binding domain is derived from a GPA. In another embodiment, a binding domain is derived from a GPA of SEQ ID NO: 55. In aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 55 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 55. In yet other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 55, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 55 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 55.

In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 55. In yet other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 55. In still other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 55.

In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 55. In yet other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 55. In still other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPA comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 55.

In an embodiment, a binding domain comprises a LIF. In another embodiment, a binding domain comprises a LIF of SEQ ID NO: 56. In another embodiment, a binding domain is derived from a LIF. In another embodiment, a binding domain is derived from a LIF of SEQ ID NO: 56. In aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 56 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 56. In yet other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 56, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 56 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 56.

In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 56. In yet other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 56. In still other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 56.

In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 56. In yet other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 56. In still other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 56.

In an embodiment, a binding domain comprises a CT-1. In another embodiment, a binding domain comprises a CT-1 of SEQ ID NO: 57. In another embodiment, a binding domain is derived from a CT-1. In another embodiment, a binding domain is derived from a CT-1 of SEQ ID NO: 57. In aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 57 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 57. In yet other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 57, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 57 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 57.

In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 57. In yet other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 57. In still other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 57.

In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 57. In yet other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 57. In still other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 57.

In an embodiment, a binding domain comprises a CLC. In another embodiment, a binding domain comprises a CLC of SEQ ID NO: 58. In another embodiment, a binding domain is derived from a CLC. In another embodiment, a binding domain is derived from a CLC of SEQ ID NO: 58. In aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 58 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 58. In yet other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 58, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 58 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 58.

In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 58. In yet other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 58. In still other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 58.

In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 58. In yet other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 58. In still other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLC comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 58.

In an embodiment, a binding domain comprises a IL-1. In another embodiment, a binding domain comprises a IL-1 of SEQ ID NO: 59. In another embodiment, a binding domain is derived from an IL-1. In another embodiment, a binding domain is derived from an IL-1 of SEQ ID NO: 59. In an aspect of this embodiment, a binding domain is derived from an IL-1 comprising amino acids 123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at least 75% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at least 80% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at least 85% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at least 90% amino acid identity with amino acids 123-265 of SEQ ID NO: 59 or at least 95% amino acid identity with amino acids 123-265 of SEQ ID NO: 59. In yet other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at most 75% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at most 80% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at most 85% amino acid identity with amino acids 123-265 of SEQ ID NO: 59, at most 90% amino acid identity with amino acids 123-265 of SEQ ID NO: 59 or at most 95% amino acid identity with amino acids 123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 123-265 of SEQ ID NO: 59. In yet other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 123-265 of SEQ ID NO: 59. In still other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 123-265 of SEQ ID NO: 59. In yet other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 123-265 of SEQ ID NO: 59. In still other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, an IL-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 123-265 of SEQ ID NO: 59.

In an embodiment, a binding domain comprises a IL-2. In another embodiment, a binding domain comprises a IL-2 of SEQ ID NO: 60. In another embodiment, a binding domain is derived from an IL-2. In another embodiment, a binding domain is derived from an IL-2 of SEQ ID NO: 60. In an aspect of this embodiment, a binding domain is derived from an IL-2 comprising amino acids 21-153 of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at least 75% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at least 80% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at least 85% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at least 90% amino acid identity with amino acids 21-153 of SEQ ID NO: 60 or at least 95% amino acid identity with amino acids 21-153 of SEQ ID NO: 60. In yet other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at most 75% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at most 80% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at most 85% amino acid identity with amino acids 21-153 of SEQ ID NO: 60, at most 90% amino acid identity with amino acids 21-153 of SEQ ID NO: 60 or at most 95% amino acid identity with amino acids 21-153 of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-153 of SEQ ID NO: 60. In yet other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-153 of SEQ ID NO: 60. In still other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-153 of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-153 of SEQ ID NO: 60. In yet other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-153 of SEQ ID NO: 60. In still other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-153 of SEQ ID NO: 60.

In an embodiment, a binding domain comprises a IL-6. In another embodiment, a binding domain comprises a IL-6 of SEQ ID NO: 61. In another embodiment, a binding domain is derived from an IL-6. In another embodiment, a binding domain is derived from an IL-6 of SEQ ID NO: 61. In an aspect of this embodiment, a binding domain is derived from an IL-6 comprising amino acids 57-210 of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at least 75% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at least 80% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at least 85% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at least 90% amino acid identity with amino acids 57-210 of SEQ ID NO: 61 or at least 95% amino acid identity with amino acids 57-210 of SEQ ID NO: 61. In yet other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at most 75% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at most 80% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at most 85% amino acid identity with amino acids 57-210 of SEQ ID NO: 61, at most 90% amino acid identity with amino acids 57-210 of SEQ ID NO: 61 or at most 95% amino acid identity with amino acids 57-210 of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 57-210 of SEQ ID NO: 61. In yet other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 57-210 of SEQ ID NO: 61. In still other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 57-210 of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 57-210 of SEQ ID NO: 61. In yet other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 57-210 of SEQ ID NO: 61. In still other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 57-210 of SEQ ID NO: 61.

In an embodiment, a binding domain comprises a IL-8. In another embodiment, a binding domain comprises a IL-8 of SEQ ID NO: 62. In another embodiment, a binding domain is derived from an IL-8. In another embodiment, a binding domain is derived from an IL-8 of SEQ ID NO: 62. In an aspect of this embodiment, a binding domain is derived from an IL-8 comprising amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at least 75% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at least 80% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at least 85% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at least 90% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62 or at least 95% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In yet other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most 75% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most 80% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most 85% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most 90% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62 or at most 95% amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In yet other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In still other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In yet other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In still other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62.

In an embodiment, a binding domain comprises a IL-10. In another embodiment, a binding domain comprises a IL-10 of SEQ ID NO: 63. In another embodiment, a binding domain is derived from an IL-10. In another embodiment, a binding domain is derived from an IL-10 of SEQ ID NO: 63. In an aspect of this embodiment, a binding domain is derived from an IL-10 comprising amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at least 75% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at least 80% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at least 85% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at least 90% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63 or at least 95% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In yet other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at most 75% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at most 80% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at most 85% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at most 90% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63 or at most 95% amino acid identity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In yet other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In still other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In yet other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In still other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In an embodiment, a binding domain comprises a neuroleukin. In another embodiment, a binding domain comprises a neuroleukin of SEQ ID NO: 64. In another embodiment, a binding domain is derived from a neuroleukin. In another embodiment, a binding domain is derived from a neuroleukin of SEQ ID NO: 64. In aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 64 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 64. In yet other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 64, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 64 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 64.

In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 64. In yet other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 64. In still other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 64.

In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 64. In yet other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 64. In still other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 64. In other aspects of this embodiment, a neuroleukin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 64.

In an embodiment, a binding domain comprises a VEGF. In another embodiment, a binding domain comprises a VEGF of SEQ ID NO: 65. In another embodiment, a binding domain is derived from a VEGF. In another embodiment, a binding domain is derived from a VEGF of SEQ ID NO: 65. In aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 65 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 65. In yet other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 65, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 65 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 65.

In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 65. In yet other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 65. In still other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 65.

In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 65. In yet other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 65. In still other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 65.

In an embodiment, a binding domain comprises a IGF-1. In another embodiment, a binding domain comprises a IGF-1 of SEQ ID NO: 66. In another embodiment, a binding domain is derived from an IGF-1. In another embodiment, a binding domain is derived from an IGF-1 of SEQ ID NO: 66. In an aspect of this embodiment, a binding domain is derived from an IGF-1 comprising amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at least 75% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at least 80% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at least 85% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at least 90% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66 or at least 95% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In yet other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at most 75% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at most 80% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at most 85% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at most 90% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66 or at most 95% amino acid identity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In yet other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In still other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In yet other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In still other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In an embodiment, a binding domain comprises a IGF-2. In another embodiment, a binding domain comprises a IGF-2 of SEQ ID NO: 67. In another embodiment, a binding domain is derived from an IGF-2. In another embodiment, a binding domain is derived from an IGF-2 of SEQ ID NO: 67. In an aspect of this embodiment, a binding domain is derived from an IGF-2 comprising amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at least 75% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at least 80% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at least 85% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at least 90% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67 or at least 95% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In yet other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at most 75% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at most 80% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at most 85% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at most 90% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67 or at most 95% amino acid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In yet other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In still other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In yet other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In still other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In an embodiment, a binding domain comprises a EGF. In another embodiment, a binding domain comprises a EGF of SEQ ID NO: 68. In another embodiment, a binding domain is derived from an EGF. In another embodiment, a binding domain is derived from an EGF of SEQ ID NO: 68. In aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at least 75% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at least 85% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 68 or at least 95% amino acid identity with the amino acid sequence of SEQ ID NO: 68. In yet other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most 70% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at most 75% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at most 80% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at most 85% amino acid identity with the amino acid sequence of SEQ ID NO: 68, at most 90% amino acid identity with the amino acid sequence of SEQ ID NO: 68 or at most 95% amino acid identity with the amino acid sequence of SEQ ID NO: 68.

In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 68. In yet other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 68. In still other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 68.

In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 68. In yet other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to the amino acid sequence of SEQ ID NO: 68. In still other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to the amino acid sequence of SEQ ID NO: 68.

Another example of a binding domain disclosed in the present specification is, e.g., a neurotrophin, such as, e.g., a NGF, a BDNF, a NT-3 or a NT-5.

Thus, in an embodiment, a binding domain comprises a NGF. In another embodiment, a binding domain comprises a NGF of SEQ ID NO: 69. In another embodiment, a binding domain is derived from a NGF. In another embodiment, a binding domain is derived from a NGF of SEQ ID NO: 69. In an aspect of this embodiment, a binding domain is derived from a NGF comprising amino acids 139-257 of SEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at least 75% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at least 80% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at least 85% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at least 90% amino acid identity with amino acids 139-257 of SEQ ID NO: 69 or at least 95% amino acid identity with amino acids 139-257 of SEQ ID NO: 69. In yet other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at most 75% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at most 80% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at most 85% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, at most 90% amino acid identity with amino acids 139-257 of SEQ ID NO: 69 or at most 95% amino acid identity with amino acids 139-257 of SEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 139-257 of SEQ ID NO: 69. In yet other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 139-257 of SEQ ID NO: 69. In still other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 139-257 of SEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 139-257 of SEQ ID NO: 69. In yet other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 139-257 of SEQ ID NO: 69. In still other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 139-257 of SEQ ID NO: 69.

In an embodiment, a binding domain comprises a BDGF. In another embodiment, a binding domain comprises a BDGF of SEQ ID NO: 70. In another embodiment, a binding domain is derived from a BDGF. In another embodiment, a binding domain is derived from a BDGF of SEQ ID NO: 70. In an aspect of this embodiment, a binding domain is derived from a BDGF comprising amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at least 75% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at least 80% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at least 85% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at least 90% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70 or at least 95% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In yet other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at most 75% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at most 80% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at most 85% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at most 90% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70 or at most 95% amino acid identity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In yet other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In still other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In yet other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In still other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In an embodiment, a binding domain comprises a NT-3. In another embodiment, a binding domain comprises a NT-3 of SEQ ID NO: 71. In another embodiment, a binding domain is derived from a NT-3. In another embodiment, a binding domain is derived from a NT-3 of SEQ ID NO: 71. In an aspect of this embodiment, a binding domain is derived from a NT-3 comprising amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at least 75% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at least 80% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at least 85% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at least 90% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71 or at least 95% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In yet other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at most 75% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at most 80% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at most 85% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at most 90% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71 or at most 95% amino acid identity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In yet other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In still other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In yet other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In still other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In an embodiment, a binding domain comprises a NT-4/5. In another embodiment, a binding domain comprises a NT-4/5 of SEQ ID NO: 72. In another embodiment, a binding domain is derived from a NT-4/5. In another embodiment, a binding domain is derived from a NT-4/5 of SEQ ID NO: 72. In an aspect of this embodiment, a binding domain is derived from a NT-4/5 comprising amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at least 75% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at least 80% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at least 85% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at least 90% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72 or at least 95% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In yet other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at most 75% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at most 80% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at most 85% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at most 90% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72 or at most 95% amino acid identity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In yet other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In still other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In yet other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In still other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

Another example of a binding domain disclosed in the present specification is, e.g., a GDNF, a neurturin, a persephrin or an artemin.

Thus, in an embodiment, a binding domain comprises a GDNF. In another embodiment, a binding domain comprises a GDNF of SEQ ID NO: 73. In another embodiment, a binding domain is derived from a GDNF. In another embodiment, a binding domain is derived from a GDNF of SEQ ID NO: 73. In an aspect of this embodiment, a binding domain is derived from a GDNF comprising amino acids 118-211 of SEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at least 75% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at least 80% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at least 85% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at least 90% amino acid identity with amino acids 118-211 of SEQ ID NO: 73 or at least 95% amino acid identity with amino acids 118-211 of SEQ ID NO: 73. In yet other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at most 75% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at most 80% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at most 85% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, at most 90% amino acid identity with amino acids 118-211 of SEQ ID NO: 73 or at most 95% amino acid identity with amino acids 118-211 of SEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 118-211 of SEQ ID NO: 73. In yet other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 118-211 of SEQ ID NO: 73. In still other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 118-211 of SEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 118-211 of SEQ ID NO: 73. In yet other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 118-211 of SEQ ID NO: 73. In still other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 118-211 of SEQ ID NO: 73.

In an embodiment, a binding domain comprises a Neurturin. In another embodiment, a binding domain comprises a Neurturin of SEQ ID NO: 74. In another embodiment, a binding domain is derived from a Neurturin. In another embodiment, a binding domain is derived from a Neurturin of SEQ ID NO: 74. In an aspect of this embodiment, a binding domain is derived from a Neurturin comprising amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at least 75% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at least 80% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at least 85% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at least 90% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74 or at least 95% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In yet other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at most 75% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at most 80% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at most 85% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at most 90% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74 or at most 95% amino acid identity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In yet other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In still other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In yet other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In still other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, a Neurturin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74.

In an embodiment, a binding domain comprises a Persephrin. In another embodiment, a binding domain comprises a Persephrin of SEQ ID NO: 75. In another embodiment, a binding domain is derived from a Persephrin. In another embodiment, a binding domain is derived from a Persephrin of SEQ ID NO: 75. In an aspect of this embodiment, a binding domain is derived from a Persephrin comprising amino acids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at least 75% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at least 80% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at least 85% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at least 90% amino acid identity with amino acids 66-155 of SEQ ID NO: 75 or at least 95% amino acid identity with amino acids 66-155 of SEQ ID NO: 75. In yet other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at most 75% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at most 80% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at most 85% amino acid identity with amino acids 66-155 of SEQ ID NO: 75, at most 90% amino acid identity with amino acids 66-155 of SEQ ID NO: 75 or at most 95% amino acid identity with amino acids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 66-155 of SEQ ID NO: 75. In yet other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 66-155 of SEQ ID NO: 75. In still other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 66-155 of SEQ ID NO: 75. In yet other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 66-155 of SEQ ID NO: 75. In still other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 66-155 of SEQ ID NO: 75. In other aspects of this embodiment, a Persephrin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 66-155 of SEQ ID NO: 75.

In an embodiment, a binding domain comprises an Artemin. In another embodiment, a binding domain comprises an Artemin of SEQ ID NO: 76. In another embodiment, a binding domain is derived from an Artemin. In another embodiment, a binding domain is derived from an Artemin of SEQ ID NO: 76. In an aspect of this embodiment, a binding domain is derived from an Artemin comprising amino acids 123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at least 75% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at least 80% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at least 85% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at least 90% amino acid identity with amino acids 123-218 of SEQ ID NO: 76 or at least 95% amino acid identity with amino acids 123-218 of SEQ ID NO: 76. In yet other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at most 75% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at most 80% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at most 85% amino acid identity with amino acids 123-218 of SEQ ID NO: 76, at most 90% amino acid identity with amino acids 123-218 of SEQ ID NO: 76 or at most 95% amino acid identity with amino acids 123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 123-218 of SEQ ID NO: 76. In yet other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 123-218 of SEQ ID NO: 76. In still other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 123-218 of SEQ ID NO: 76. In yet other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 123-218 of SEQ ID NO: 76. In still other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, an Artemin comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 123-218 of SEQ ID NO: 76.

Another example of a binding domain disclosed in the present specification is, e.g., a TGFβs, such as, e.g., TGFβ1, TGFβ2, TGFβ3 or TGFβ4.

Thus, in an embodiment, a binding domain comprises a TGFβ1. In another embodiment, a binding domain comprises a TGFβ1 of SEQ ID NO: 77. In another embodiment, a binding domain is derived from a TGFβ1. In another embodiment, a binding domain is derived from a TGFβ1 of SEQ ID NO: 77. In an aspect of this embodiment, a binding domain is derived from a TGFβ1 comprising amino acids 293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at least 75% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at least 80% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at least 85% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at least 90% amino acid identity with amino acids 293-390 of SEQ ID NO: 77 or at least 95% amino acid identity with amino acids 293-390 of SEQ ID NO: 77. In yet other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at most 75% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at most 80% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at most 85% amino acid identity with amino acids 293-390 of SEQ ID NO: 77, at most 90% amino acid identity with amino acids 293-390 of SEQ ID NO: 77 or at most 95% amino acid identity with amino acids 293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 293-390 of SEQ ID NO: 77. In yet other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 293-390 of SEQ ID NO: 77. In still other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 293-390 of SEQ ID NO: 77. In yet other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 293-390 of SEQ ID NO: 77. In still other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 293-390 of SEQ ID NO: 77.

In an embodiment, a binding domain comprises a TGFβ2. In another embodiment, a binding domain comprises a TGFβ2 of SEQ ID NO: 78. In another embodiment, a binding domain is derived from a TGFβ2. In another embodiment, a binding domain is derived from a TGFβ2 of SEQ ID NO: 77. In an aspect of this embodiment, a binding domain is derived from a TGFβ2 comprising amino acids 317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at least 75% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at least 80% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at least 85% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at least 90% amino acid identity with amino acids 317-414 of SEQ ID NO: 78 or at least 95% amino acid identity with amino acids 317-414 of SEQ ID NO: 78. In yet other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at most 75% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at most 80% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at most 85% amino acid identity with amino acids 317-414 of SEQ ID NO: 78, at most 90% amino acid identity with amino acids 317-414 of SEQ ID NO: 78 or at most 95% amino acid identity with amino acids 317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 317-414 of SEQ ID NO: 78. In yet other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 317-414 of SEQ ID NO: 78. In still other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 317-414 of SEQ ID NO: 78. In yet other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 317-414 of SEQ ID NO: 78. In still other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 317-414 of SEQ ID NO: 78.

In an embodiment, a binding domain comprises a TGFβ3. In another embodiment, a binding domain comprises a TGFβ3 of SEQ ID NO: 79. In another embodiment, a binding domain is derived from a TGFβ3. In another embodiment, a binding domain is derived from a TGFβ3 of SEQ ID NO: 77. In an aspect of this embodiment, a binding domain is derived from a TGFβ3 comprising amino acids 315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at least 75% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at least 80% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at least 85% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at least 90% amino acid identity with amino acids 315-412 of SEQ ID NO: 79 or at least 95% amino acid identity with amino acids 315-412 of SEQ ID NO: 79. In yet other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at most 75% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at most 80% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at most 85% amino acid identity with amino acids 315-412 of SEQ ID NO: 79, at most 90% amino acid identity with amino acids 315-412 of SEQ ID NO: 79 or at most 95% amino acid identity with amino acids 315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 315-412 of SEQ ID NO: 79. In yet other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 315-412 of SEQ ID NO: 79. In still other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 315-412 of SEQ ID NO: 79. In yet other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 315-412 of SEQ ID NO: 79. In still other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 315-412 of SEQ ID NO: 79.

In an embodiment, a binding domain comprises a TGFβ4. In another embodiment, a binding domain comprises a TGFβ4 of SEQ ID NO: 80. In another embodiment, a binding domain is derived from a TGFβ4. In another embodiment, a binding domain is derived from a TGFβ4 of SEQ ID NO: 77. In an aspect of this embodiment, a binding domain is derived from a TGFβ4 comprising amino acids 276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at least 75% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at least 80% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at least 85% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at least 90% amino acid identity with amino acids 276-373 of SEQ ID NO: 80 or at least 95% amino acid identity with amino acids 276-373 of SEQ ID NO: 80. In yet other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at most 75% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at most 80% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at most 85% amino acid identity with amino acids 276-373 of SEQ ID NO: 80, at most 90% amino acid identity with amino acids 276-373 of SEQ ID NO: 80 or at most 95% amino acid identity with amino acids 276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 276-373 of SEQ ID NO: 80. In yet other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 276-373 of SEQ ID NO: 80. In still other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 276-373 of SEQ ID NO: 80. In yet other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 276-373 of SEQ ID NO: 80. In still other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 276-373 of SEQ ID NO: 80.

Another example of a binding domain disclosed in the present specification is, e.g., a BMPs, such as, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8 or BMP10.

Thus, in an embodiment, a binding domain comprises a BMP2. In another embodiment, a binding domain comprises a BMP2 of SEQ ID NO: 81. In another embodiment, a binding domain is derived from a BMP2. In another embodiment, a binding domain is derived from a BMP2 of SEQ ID NO: 81. In an aspect of this embodiment, a binding domain is derived from a BMP2 comprising amino acids 296-396 of SEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at least 75% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at least 80% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at least 85% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at least 90% amino acid identity with amino acids 296-396 of SEQ ID NO: 81 or at least 95% amino acid identity with amino acids 296-396 of SEQ ID NO: 81. In yet other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at most 75% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at most 80% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at most 85% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, at most 90% amino acid identity with amino acids 296-396 of SEQ ID NO: 81 or at most 95% amino acid identity with amino acids 296-396 of SEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 296-396 of SEQ ID NO: 81. In yet other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 296-396 of SEQ ID NO: 81. In still other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 296-396 of SEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 296-396 of SEQ ID NO: 81. In yet other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 296-396 of SEQ ID NO: 81. In still other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 296-396 of SEQ ID NO: 81.

In an embodiment, a binding domain comprises a BMP3. In another embodiment, a binding domain comprises a BMP3 of SEQ ID NO: 82. In another embodiment, a binding domain is derived from a BMP3. In another embodiment, a binding domain is derived from a BMP3 of SEQ ID NO: 82. In an aspect of this embodiment, a binding domain is derived from a BMP3 comprising amino acids 370-472 of SEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at least 75% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at least 80% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at least 85% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at least 90% amino acid identity with amino acids 370-472 of SEQ ID NO: 82 or at least 95% amino acid identity with amino acids 370-472 of SEQ ID NO: 82. In yet other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at most 75% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at most 80% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at most 85% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, at most 90% amino acid identity with amino acids 370-472 of SEQ ID NO: 82 or at most 95% amino acid identity with amino acids 370-472 of SEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 370-472 of SEQ ID NO: 82. In yet other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 370-472 of SEQ ID NO: 82. In still other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 370-472 of SEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 370-472 of SEQ ID NO: 82. In yet other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 370-472 of SEQ ID NO: 82. In still other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 370-472 of SEQ ID NO: 82.

In an embodiment, a binding domain comprises a BMP4. In another embodiment, a binding domain comprises a BMP4 of SEQ ID NO: 83. In another embodiment, a binding domain is derived from a BMP4. In another embodiment, a binding domain is derived from a BMP4 of SEQ ID NO: 83. In an aspect of this embodiment, a binding domain is derived from a BMP4 comprising amino acids 309-409 of SEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at least 75% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at least 80% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at least 85% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at least 90% amino acid identity with amino acids 309-409 of SEQ ID NO: 83 or at least 95% amino acid identity with amino acids 309-409 of SEQ ID NO: 83. In yet other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at most 75% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at most 80% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at most 85% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, at most 90% amino acid identity with amino acids 309-409 of SEQ ID NO: 83 or at most 95% amino acid identity with amino acids 309-409 of SEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 309-409 of SEQ ID NO: 83. In yet other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 309-409 of SEQ ID NO: 83. In still other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 309-409 of SEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 309-409 of SEQ ID NO: 83. In yet other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 309-409 of SEQ ID NO: 83. In still other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 309-409 of SEQ ID NO: 83.

In an embodiment, a binding domain comprises a BMP5. In another embodiment, a binding domain comprises a BMP5 of SEQ ID NO: 84. In another embodiment, a binding domain is derived from a BMP5. In another embodiment, a binding domain is derived from a BMP5 of SEQ ID NO: 84. In an aspect of this embodiment, a binding domain is derived from a BMP5 comprising amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at least 75% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at least 80% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at least 85% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at least 90% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84 or at least 95% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In yet other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at most 75% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at most 80% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at most 85% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at most 90% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84 or at most 95% amino acid identity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In yet other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In still other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In yet other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In still other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In an embodiment, a binding domain comprises a BMP6. In another embodiment, a binding domain comprises a BMP6 of SEQ ID NO: 85. In another embodiment, a binding domain is derived from a BMP6. In another embodiment, a binding domain is derived from a BMP6 of SEQ ID NO: 85. In an aspect of this embodiment, a binding domain is derived from a BMP6 comprising amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at least 75% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at least 80% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at least 85% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at least 90% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85 or at least 95% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In yet other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at most 75% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at most 80% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at most 85% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at most 90% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85 or at most 95% amino acid identity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In yet other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In still other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In yet other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In still other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In an embodiment, a binding domain comprises a BMP7. In another embodiment, a binding domain comprises a BMP7 of SEQ ID NO: 86. In another embodiment, a binding domain is derived from a BMP7. In another embodiment, a binding domain is derived from a BMP7 of SEQ ID NO: 86. In an aspect of this embodiment, a binding domain is derived from a BMP7 comprising amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at least 75% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at least 80% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at least 85% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at least 90% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86 or at least 95% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In yet other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at most 75% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at most 80% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at most 85% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at most 90% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86 or at most 95% amino acid identity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In yet other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In still other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In yet other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In still other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In an embodiment, a binding domain comprises a BMP8. In another embodiment, a binding domain comprises a BMP8 of SEQ ID NO: 87. In another embodiment, a binding domain is derived from a BMP8. In another embodiment, a binding domain is derived from a BMP8 of SEQ ID NO: 87. In an aspect of this embodiment, a binding domain is derived from a BMP8 comprising amino acids 301-402 of SEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at least 75% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at least 80% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at least 85% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at least 90% amino acid identity with amino acids 301-402 of SEQ ID NO: 87 or at least 95% amino acid identity with amino acids 301-402 of SEQ ID NO: 87. In yet other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at most 75% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at most 80% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at most 85% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, at most 90% amino acid identity with amino acids 301-402 of SEQ ID NO: 87 or at most 95% amino acid identity with amino acids 301-402 of SEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 301-402 of SEQ ID NO: 87. In yet other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 301-402 of SEQ ID NO: 87. In still other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 301-402 of SEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 301-402 of SEQ ID NO: 87. In yet other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 301-402 of SEQ ID NO: 87. In still other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 301-402 of SEQ ID NO: 87.

In an embodiment, a binding domain comprises a BMP10. In another embodiment, a binding domain comprises a BMP10 of SEQ ID NO: 88. In another embodiment, a binding domain is derived from a BMP10. In another embodiment, a binding domain is derived from a BMP10 of SEQ ID NO: 88. In an aspect of this embodiment, a binding domain is derived from a BMP10 comprising amino acids 323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at least 75% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at least 80% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at least 85% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at least 90% amino acid identity with amino acids 323-424 of SEQ ID NO: 88 or at least 95% amino acid identity with amino acids 323-424 of SEQ ID NO: 88. In yet other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at most 75% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at most 80% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at most 85% amino acid identity with amino acids 323-424 of SEQ ID NO: 88, at most 90% amino acid identity with amino acids 323-424 of SEQ ID NO: 88 or at most 95% amino acid identity with amino acids 323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 323-424 of SEQ ID NO: 88. In yet other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 323-424 of SEQ ID NO: 88. In still other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 323-424 of SEQ ID NO: 88. In yet other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 323-424 of SEQ ID NO: 88. In still other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 323-424 of SEQ ID NO: 88.

Another example of a binding domain disclosed in the present specification is, e.g., a GFPs, such as, e.g., GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF10, GDF11 or GDF15.

Thus, in an embodiment, a binding domain comprises a GDF1. In another embodiment, a binding domain comprises a GDF1 of SEQ ID NO: 89. In another embodiment, a binding domain is derived from a GDF1. In another embodiment, a binding domain is derived from a GDF1 of SEQ ID NO: 89. In an aspect of this embodiment, a binding domain is derived from a GDF1 comprising amino acids 267-372 of SEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at least 75% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at least 80% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at least 85% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at least 90% amino acid identity with amino acids 267-372 of SEQ ID NO: 89 or at least 95% amino acid identity with amino acids 267-372 of SEQ ID NO: 89. In yet other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at most 75% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at most 80% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at most 85% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, at most 90% amino acid identity with amino acids 267-372 of SEQ ID NO: 89 or at most 95% amino acid identity with amino acids 267-372 of SEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 267-372 of SEQ ID NO: 89. In yet other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 267-372 of SEQ ID NO: 89. In still other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 267-372 of SEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 267-372 of SEQ ID NO: 89. In yet other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 267-372 of SEQ ID NO: 89. In still other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 267-372 of SEQ ID NO: 89.

In an embodiment, a binding domain comprises a GDF2. In another embodiment, a binding domain comprises a GDF2 of SEQ ID NO: 90. In another embodiment, a binding domain is derived from a GDF2. In another embodiment, a binding domain is derived from a GDF2 of SEQ ID NO: 90. In an aspect of this embodiment, a binding domain is derived from a GDF2 comprising amino acids 327-429 of SEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at least 75% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at least 80% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at least 85% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at least 90% amino acid identity with amino acids 327-429 of SEQ ID NO: 90 or at least 95% amino acid identity with amino acids 327-429 of SEQ ID NO: 90. In yet other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at most 75% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at most 80% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at most 85% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, at most 90% amino acid identity with amino acids 327-429 of SEQ ID NO: 90 or at most 95% amino acid identity with amino acids 327-429 of SEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 327-429 of SEQ ID NO: 90. In yet other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 327-429 of SEQ ID NO: 90. In still other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 327-429 of SEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 327-429 of SEQ ID NO: 90. In yet other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 327-429 of SEQ ID NO: 90. In still other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 327-429 of SEQ ID NO: 90.

In an embodiment, a binding domain comprises a GDF3. In another embodiment, a binding domain comprises a GDF3 of SEQ ID NO: 91. In another embodiment, a binding domain is derived from a GDF3. In another embodiment, a binding domain is derived from a GDF3 of SEQ ID NO: 91. In an aspect of this embodiment, a binding domain is derived from a GDF3 comprising amino acids 264-364 of SEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at least 75% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at least 80% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at least 85% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at least 90% amino acid identity with amino acids 264-364 of SEQ ID NO: 91 or at least 95% amino acid identity with amino acids 264-364 of SEQ ID NO: 91. In yet other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at most 75% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at most 80% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at most 85% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, at most 90% amino acid identity with amino acids 264-364 of SEQ ID NO: 91 or at most 95% amino acid identity with amino acids 264-364 of SEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 264-364 of SEQ ID NO: 91. In yet other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 264-364 of SEQ ID NO: 91. In still other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 264-364 of SEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 264-364 of SEQ ID NO: 91. In yet other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 264-364 of SEQ ID NO: 91. In still other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 264-364 of SEQ ID NO: 91.

In an embodiment, a binding domain comprises a GDF5. In another embodiment, a binding domain comprises a GDF5 of SEQ ID NO: 92. In another embodiment, a binding domain is derived from a GDF5. In another embodiment, a binding domain is derived from a GDF5 of SEQ ID NO: 92. In an aspect of this embodiment, a binding domain is derived from a GDF5 comprising amino acids 400-501 of SEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at least 75% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at least 80% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at least 85% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at least 90% amino acid identity with amino acids 400-501 of SEQ ID NO: 92 or at least 95% amino acid identity with amino acids 400-501 of SEQ ID NO: 92. In yet other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at most 75% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at most 80% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at most 85% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, at most 90% amino acid identity with amino acids 400-501 of SEQ ID NO: 92 or at most 95% amino acid identity with amino acids 400-501 of SEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 400-501 of SEQ ID NO: 92. In yet other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 400-501 of SEQ ID NO: 92. In still other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 400-501 of SEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 400-501 of SEQ ID NO: 92. In yet other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 400-501 of SEQ ID NO: 92. In still other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 400-501 of SEQ ID NO: 92.

In an embodiment, a binding domain comprises a GDF6. In another embodiment, a binding domain comprises a GDF6 of SEQ ID NO: 93. In another embodiment, a binding domain is derived from a GDF6. In another embodiment, a binding domain is derived from a GDF6 of SEQ ID NO: 93. In an aspect of this embodiment, a binding domain is derived from a GDF6 comprising amino acids 354-455 of SEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at least 75% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at least 80% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at least 85% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at least 90% amino acid identity with amino acids 354-455 of SEQ ID NO: 93 or at least 95% amino acid identity with amino acids 354-455 of SEQ ID NO: 93. In yet other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at most 75% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at most 80% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at most 85% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, at most 90% amino acid identity with amino acids 354-455 of SEQ ID NO: 93 or at most 95% amino acid identity with amino acids 354-455 of SEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 354-455 of SEQ ID NO: 93. In yet other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 354-455 of SEQ ID NO: 93. In still other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 354-455 of SEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 354-455 of SEQ ID NO: 93. In yet other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 354-455 of SEQ ID NO: 93. In still other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 354-455 of SEQ ID NO: 93.

In an embodiment, a binding domain comprises a GDF7. In another embodiment, a binding domain comprises a GDF7 of SEQ ID NO: 94. In another embodiment, a binding domain is derived from a GDF7. In another embodiment, a binding domain is derived from a GDF7 of SEQ ID NO: 94. In an aspect of this embodiment, a binding domain is derived from a GDF7 comprising amino acids 352-450 of SEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at least 75% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at least 80% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at least 85% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at least 90% amino acid identity with amino acids 352-450 of SEQ ID NO: 94 or at least 95% amino acid identity with amino acids 352-450 of SEQ ID NO: 94. In yet other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at most 75% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at most 80% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at most 85% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, at most 90% amino acid identity with amino acids 352-450 of SEQ ID NO: 94 or at most 95% amino acid identity with amino acids 352-450 of SEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 352-450 of SEQ ID NO: 94. In yet other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 352-450 of SEQ ID NO: 94. In still other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 352-450 of SEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 352-450 of SEQ ID NO: 94. In yet other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 352-450 of SEQ ID NO: 94. In still other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 352-450 of SEQ ID NO: 94.

In an embodiment, a binding domain comprises a GDF8. In another embodiment, a binding domain comprises a GDF8 of SEQ ID NO: 95. In another embodiment, a binding domain is derived from a GDF8. In another embodiment, a binding domain is derived from a GDF8 of SEQ ID NO: 95. In an aspect of this embodiment, a binding domain is derived from a GDF8 comprising amino acids 281-375 of SEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at least 75% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at least 80% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at least 85% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at least 90% amino acid identity with amino acids 281-375 of SEQ ID NO: 95 or at least 95% amino acid identity with amino acids 281-375 of SEQ ID NO: 95. In yet other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at most 75% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at most 80% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at most 85% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, at most 90% amino acid identity with amino acids 281-375 of SEQ ID NO: 95 or at most 95% amino acid identity with amino acids 281-375 of SEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 281-375 of SEQ ID NO: 95. In yet other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 281-375 of SEQ ID NO: 95. In still other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 281-375 of SEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 281-375 of SEQ ID NO: 95. In yet other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 281-375 of SEQ ID NO: 95. In still other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 281-375 of SEQ ID NO: 95.

In an embodiment, a binding domain comprises a GDF10. In another embodiment, a binding domain comprises a GDF19 of SEQ ID NO: 96. In another embodiment, a binding domain is derived from a GDF10. In another embodiment, a binding domain is derived from a GDF10 of SEQ ID NO: 96. In an aspect of this embodiment, a binding domain is derived from a GDF10 comprising amino acids 376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at least 75% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at least 80% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at least 85% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at least 90% amino acid identity with amino acids 376-478 of SEQ ID NO: 96 or at least 95% amino acid identity with amino acids 376-478 of SEQ ID NO: 96. In yet other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at most 75% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at most 80% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at most 85% amino acid identity with amino acids 376-478 of SEQ ID NO: 96, at most 90% amino acid identity with amino acids 376-478 of SEQ ID NO: 96 or at most 95% amino acid identity with amino acids 376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 376-478 of SEQ ID NO: 96. In yet other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 376-478 of SEQ ID NO: 96. In still other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 376-478 of SEQ ID NO: 96. In yet other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 376-478 of SEQ ID NO: 96. In still other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 376-478 of SEQ ID NO: 96.

In an embodiment, a binding domain comprises a GDF11. In another embodiment, a binding domain comprises a GDF11 of SEQ ID NO: 97. In another embodiment, a binding domain is derived from a GDF11. In another embodiment, a binding domain is derived from a GDF11 of SEQ ID NO: 97. In an aspect of this embodiment, a binding domain is derived from a GDF11 comprising amino acids 313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at least 75% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at least 80% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at least 85% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at least 90% amino acid identity with amino acids 313-407 of SEQ ID NO: 97 or at least 95% amino acid identity with amino acids 313-407 of SEQ ID NO: 97. In yet other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at most 75% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at most 80% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at most 85% amino acid identity with amino acids 313-407 of SEQ ID NO: 97, at most 90% amino acid identity with amino acids 313-407 of SEQ ID NO: 97 or at most 95% amino acid identity with amino acids 313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 313-407 of SEQ ID NO: 97. In yet other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 313-407 of SEQ ID NO: 97. In still other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 313-407 of SEQ ID NO: 97. In yet other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 313-407 of SEQ ID NO: 97. In still other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 313-407 of SEQ ID NO: 97.

In an embodiment, a binding domain comprises a GDF15. In another embodiment, a binding domain comprises a GDF15 of SEQ ID NO: 98. In another embodiment, a binding domain is derived from a GDF15. In another embodiment, a binding domain is derived from a GDF15 of SEQ ID NO: 98. In an aspect of this embodiment, a binding domain is derived from a GDF15 comprising amino acids 211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at least 75% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at least 80% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at least 85% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at least 90% amino acid identity with amino acids 211-308 of SEQ ID NO: 98 or at least 95% amino acid identity with amino acids 211-308 of SEQ ID NO: 98. In yet other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at most 75% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at most 80% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at most 85% amino acid identity with amino acids 211-308 of SEQ ID NO: 98, at most 90% amino acid identity with amino acids 211-308 of SEQ ID NO: 98 or at most 95% amino acid identity with amino acids 211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 211-308 of SEQ ID NO: 98. In yet other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 211-308 of SEQ ID NO: 98. In still other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 211-308 of SEQ ID NO: 98. In yet other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 211-308 of SEQ ID NO: 98. In still other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15 comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 211-308 of SEQ ID NO: 98.

Another example of a binding domain disclosed in the present specification is, e.g., an activin A, an activin B, an activin C, an activin E or an inhibin A.

Thus. in an embodiment, a binding domain comprises an Activin A. In another embodiment, a binding domain comprises an Activin A of SEQ ID NO: 99. In another embodiment, a binding domain is derived from an Activin A. In another embodiment, a binding domain is derived from an Activin A of SEQ ID NO: 99. In an aspect of this embodiment, a binding domain is derived from an Activin A comprising amino acids 321-426 of SEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at least 75% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at least 80% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at least 85% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at least 90% amino acid identity with amino acids 321-426 of SEQ ID NO: 99 or at least 95% amino acid identity with amino acids 321-426 of SEQ ID NO: 99. In yet other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at most 75% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at most 80% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at most 85% amino acid identity with amino acids 321-426 of SEQ ID NO: 99, at most 90% amino acid identity with amino acids 321-426 of SEQ ID NO: 99 or at most 95% amino acid identity with amino acids 321-426 of SEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 321-426 of SEQ ID NO: 99. In yet other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 321-426 of SEQ ID NO: 99. In still other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 321-426 of SEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 321-426 of SEQ ID NO: 99. In yet other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 321-426 of SEQ ID NO: 99. In still other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 321-426 of SEQ ID NO: 99. In other aspects of this embodiment, an Activin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 321-426 of SEQ ID NO: 99.

In an embodiment, a binding domain comprises an Activin B. In another embodiment, a binding domain comprises an Activin B of SEQ ID NO: 100. In another embodiment, a binding domain is derived from an Activin B. In another embodiment, a binding domain is derived from an Activin B of SEQ ID NO: 100. In an aspect of this embodiment, a binding domain is derived from an Activin B comprising amino acids 303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at least 75% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at least 80% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at least 85% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at least 90% amino acid identity with amino acids 303-406 of SEQ ID NO: 100 or at least 95% amino acid identity with amino acids 303-406 of SEQ ID NO: 100. In yet other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at most 75% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at most 80% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at most 85% amino acid identity with amino acids 303-406 of SEQ ID NO: 100, at most 90% amino acid identity with amino acids 303-406 of SEQ ID NO: 100 or at most 95% amino acid identity with amino acids 303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 303-406 of SEQ ID NO: 100. In yet other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 303-406 of SEQ ID NO: 100. In still other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 303-406 of SEQ ID NO: 100. In yet other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 303-406 of SEQ ID NO: 100. In still other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 303-406 of SEQ ID NO: 100. In other aspects of this embodiment, an Activin B comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 303-406 of SEQ ID NO: 100.

In an embodiment, a binding domain comprises an Activin C. In another embodiment, a binding domain comprises an Activin C of SEQ ID NO: 101. In another embodiment, a binding domain is derived from an Activin C. In another embodiment, a binding domain is derived from an Activin C of SEQ ID NO: 101. In an aspect of this embodiment, a binding domain is derived from an Activin C comprising amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101.

In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 75% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 80% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 85% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 90% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101 or at least 95% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In yet other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at most 75% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at most 80% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at most 85% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at most 90% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101 or at most 95% amino acid identity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101.

In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In yet other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In still other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101.

In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In yet other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In still other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, an Activin C comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101.

In an embodiment, a binding domain comprises an Activin D. In another embodiment, a binding domain comprises an Activin D of SEQ ID NO: 102. In another embodiment, a binding domain is derived from an Activin E. In another embodiment, a binding domain is derived from an Activin E of SEQ ID NO: 102. In an aspect of this embodiment, a binding domain is derived from an Activin E comprising amino acids 247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at least 75% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at least 80% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at least 85% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at least 90% amino acid identity with amino acids 247-350 of SEQ ID NO: 102 or at least 95% amino acid identity with amino acids 247-350 of SEQ ID NO: 102. In yet other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at most 75% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at most 80% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at most 85% amino acid identity with amino acids 247-350 of SEQ ID NO: 102, at most 90% amino acid identity with amino acids 247-350 of SEQ ID NO: 102 or at most 95% amino acid identity with amino acids 247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 247-350 of SEQ ID NO: 102. In yet other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 247-350 of SEQ ID NO: 102. In still other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 247-350 of SEQ ID NO: 102. In yet other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 247-350 of SEQ ID NO: 102. In still other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 247-350 of SEQ ID NO: 102. In other aspects of this embodiment, an Activin E comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 247-350 of SEQ ID NO: 102.

In an embodiment, a binding domain comprises an Inhibin A. In another embodiment, a binding domain comprises an Inhibin A of SEQ ID NO: 103. In another embodiment, a binding domain is derived from an Inhibin A. In another embodiment, a binding domain is derived from an Inhibin A of SEQ ID NO: 103. In an aspect of this embodiment, a binding domain is derived from an Inhibin A comprising amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103.

In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 75% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 80% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 85% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 90% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103 or at least 95% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In yet other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at most 75% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at most 80% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at most 85% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at most 90% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103 or at most 95% amino acid identity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103.

In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid substitutions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In yet other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid deletions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In still other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acid additions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103.

In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid substitutions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In yet other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In still other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, an Inhibin A comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10 or 20 contiguous amino acid additions relative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103.

Another example of a binding domain includes, without limitation, a opioid peptide, such as, e.g., an enkephalin, an endomorphin, an endorphin, a dynorphin, a nociceptin or a hemorphin. Thus, in an embodiment, a binding domain comprises an opioid peptide. In another embodiment, a binding domain is derived from an opioid peptide.

In another embodiment, an opioid binding domain comprises an enkephalin peptide. In aspects of this embodiment, an opioid binding domain is derived from an enkephalin peptide. In other aspects of this embodiment, an enkephalin binding domain is derived from a Leu-enkephalin, a Met-enkephalin, a Met-enkephalin MRGL or a Met-enkephalin MRF. In other aspects of this embodiment, an enkephalin binding domain comprises SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at least 75% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at least 80% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at least 85% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at least 90% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107 or at least 95% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 75% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 80% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 85% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 90% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107 or at most 95% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid substitutions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid substitutions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid deletions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid deletions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In still other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid additions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid additions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid substitutions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid substitutions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid deletions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid deletions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In still other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid additions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of this embodiment, an enkephalin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid additions relative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In another embodiment, an opioid binding domain comprises a bovine adrenomedullary-22 (BAM22) peptide. In aspects of this embodiment, an opioid binding domain comprising a BAM22 peptide is derived from a BAM22 peptide (1-12), a BAM22 peptide (6-22), a BAM22 peptide (8-22) or a BAM22 peptide (1-22). In other aspects of this embodiment, a BAM22 binding domain comprises amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 75% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 80% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 85% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 90% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113 or at least 95% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most 75% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most 80% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most 85% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most 90% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113 or at most 95% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In still other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In still other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In another embodiment, an opioid binding domain comprises an endomorphin peptide. In another embodiment, an opioid binding domain is derived from an endomorphin peptide. In aspects of this embodiment, an endomorphin binding domain comprises an endomorphin-1 or an endomorphin-2. In other aspects of this embodiment, an endomorphin binding domain comprises SEQ ID NO: 114 or SEQ ID NO: 115.

In other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at least 75% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at least 80% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at least 85% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at least 90% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115 or at least 95% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at most 75% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at most 80% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at most 85% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115, at most 90% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115 or at most 95% amino acid identity with SEQ ID NO: 114 or SEQ ID NO: 115.

In other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In still other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three non-contiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three non-contiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO: 115.

In other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In still other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at least one, two or three contiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of this embodiment, an endomorphin binding domain comprises a polypeptide having, e.g., at most one, two or three contiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO: 115.

In another embodiment, an opioid binding domain comprises an endorphin peptide. In aspects of this embodiment, an opioid binding domain comprises is derived from an endorphin peptide. In other aspects, an endorphin binding domain comprises an endorphin-α, a neoendorphin-α, an endorphin-β, a neoendorphin-β or an endorphin-γ. In other aspects of this embodiment, an endorphin binding domain comprises SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 75% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 80% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 85% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 90% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120 or at least 95% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 75% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 80% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 85% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 90% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120 or at most 95% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In still other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In still other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In another embodiment, an opioid binding domain comprises a dynorphin peptide. In another embodiment, an opioid binding domain is derived from a dynorphin peptide. In aspects of this embodiment, a dynorphin binding domain comprises a dynorphin A, a dynorphin B (leumorphin) or a rimorphin. In other aspects of this embodiment, a dynorphin binding domain comprises SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 or SEQ ID NO: 151.

In other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 75% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 80% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 85% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 90% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146 or at least 95% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at most 75% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at most 80% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at most 85% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at most 90% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146 or at most 95% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146.

In other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In still other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146.

In other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In still other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, a dynorphin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146.

In another embodiment, an opioid binding domain comprises a nociceptin peptide. In another embodiment, an opioid binding domain is derived from a nociceptin peptide. In aspects of this embodiment, a nociceptin binding domain comprises a nociceptin RK, a nociceptin, a neuropeptide 1, a neuropeptide 2 or a neuropeptide 3. In other aspects of this embodiment, a nociceptin binding domain comprises SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at least 75% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at least 80% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at least 85% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at least 90% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161 or at least 95% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 75% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 80% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 85% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 90% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161 or at most 95% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In still other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In still other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

Another example of a binding domain disclosed in the present specification is, e.g., a melanocortin peptide, such as, e.g., a melanocyte stimulating hormone, an adrenocorticotropin, a Corticotropin-like intermediary peptide) or a lipotropin. Thus, in an embodiment, a binding domain is derived from a melanocortin peptide.

In another embodiment, a binding domain comprises a melanocortin peptide. In another embodiment, a binding domain is derived from a melanocortin peptide. In an aspect of this embodiment, a melanocortin peptide binding domain comprises a melanocyte stimulating hormone. In an aspect of this embodiment, a melanocortin peptide binding domain comprises is derived from a melanocyte stimulating hormone. In aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises an α-melanocyte stimulating hormones (α-MSH), a β-melanocyte stimulating hormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH). In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at least 75% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at least 80% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at least 85% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at least 90% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164 or at least 95% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most 75% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most 80% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most 85% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most 90% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164 or at most 95% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In still other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In still other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, a melanocyte stimulating hormone binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164.

In another embodiment, a melanocortin peptide binding domain comprises an adrenocorticotropin. In another embodiment, a melanocortin peptide binding domain is derived from an adrenocorticotropin. In aspects of this embodiment, an adrenocorticotropin binding domain comprises an adrenocorticotropin (ACTH) or a Corticotropin-like intermediary peptide (CLIP). In other aspects of this embodiment, an adrenocorticotropin binding domain comprises SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 75% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 80% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 85% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 90% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166 or at least 95% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at most 75% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at most 80% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at most 85% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166, at most 90% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166 or at most 95% amino acid identity with SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In still other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In still other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet other aspects of this embodiment, an adrenocorticotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 165 or SEQ ID NO: 166.

In another embodiment, a melanocortin peptide binding domain comprises a lipotropin. In another embodiment, a melanocortin peptide binding domain is derived from a lipotropin. In aspects of this embodiment, a lipotropin binding domain comprises a β-lipotropin (β-LPH) or a γ-lipotropin (γ-LPH). In other aspects of this embodiment, a lipotropin binding domain comprises SEQ ID NO: 167 or SEQ ID NO: 168.

In other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at least 75% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at least 80% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at least 85% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at least 90% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168 or at least 95% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at most 75% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at most 80% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at most 85% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168, at most 90% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168 or at most 95% amino acid identity with SEQ ID NO: 167 or SEQ ID NO: 168.

In other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In still other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 167 or SEQ ID NO: 168.

In other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In still other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 167 or SEQ ID NO: 168.

In another embodiment, a melanocortin peptide binding domain comprises a neuropeptide derived from a melanocortin peptide. In another embodiment, a melanocortin peptide binding domain is derived from a neuropeptide derived from a melanocortin peptide. In aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least 75% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least 80% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least 85% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least 90% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171 or at least 95% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most 75% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most 80% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most 85% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most 90% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171 or at most 95% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In still other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In still other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, a melanocortin peptide derived neuropeptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In another embodiment, a binding domain comprises a galanin. In another embodiment, a binding domain is derived from a galanin. In aspects of this embodiment, a galanin binding domain comprises a galanin or a galanin message-associated peptide (GMAP). In other aspects of this embodiment, a galanin binding domain comprises SEQ ID NO: 172 or SEQ ID NO: 173.

In other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at least 75% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at least 80% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at least 85% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at least 90% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173 or at least 95% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 75% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 80% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 85% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 90% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173 or at most 95% amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173.

In other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In still other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 172 or SEQ ID NO: 173.

In other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In still other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, a galanin binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 172 or SEQ ID NO: 173.

Another example of a binding domain disclosed in the present specification is, e.g., a granin peptide, such as, e.g., a chromogranin A, a chromogranin B (secretogranin 1) or a chromogranin C (secretogranin II). Thus, in an embodiment, a binding domain comprises a granin peptide. In another embodiment, a binding domain is derived from a granin peptide.

In another embodiment, a granin peptide binding domain comprises a chromogranin A peptide. In another embodiment, a granin peptide binding domain is derived from a chromogranin A peptide. In aspects of this embodiment, a chromogranin A peptide binding domain comprises a β-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, a catestatin, a parastatin or a GE-25. In other aspects of this embodiment, a chromogranin A peptide binding domain comprises SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at least 75% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at least 80% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at least 85% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at least 90% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 or at least 95% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 75% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 80% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 85% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 90% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 or at most 95% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In still other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In still other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a chromogranin A peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In another embodiment, a granin peptide binding domain comprises a chromogranin B peptide. In another embodiment, a granin peptide binding domain is derived from a chromogranin B peptide. In aspects of this embodiment, a chromogranin B peptide binding domain comprises a GAWK peptide, an adrenomedullary peptide or a secretolytin. In other aspects of this embodiment, a chromogranin B peptide binding domain comprises SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186.

In other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at least 75% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at least 80% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at least 85% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at least 90% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186 or at least 95% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at most 75% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at most 80% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at most 85% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at most 90% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186 or at most 95% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186.

In other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In still other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186.

In other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In still other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of this embodiment, a chromogranin B peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186.

In another embodiment, a granin peptide binding domain comprises a chromogranin C peptide. In another embodiment, a granin peptide binding domain is derived from a chromogranin C peptide. In aspects of this embodiment, a chromogranin C peptide binding domain comprises a secretoneurin. In other aspects of this embodiment, a chromogranin C peptide binding domain comprises SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 187, at least 75% amino acid identity with SEQ ID NO: 187, at least 80% amino acid identity with SEQ ID NO: 187, at least 85% amino acid identity with SEQ ID NO: 187, at least 90% amino acid identity with SEQ ID NO: 187 or at least 95% amino acid identity with SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 187, at most 75% amino acid identity with SEQ ID NO: 187, at most 80% amino acid identity with SEQ ID NO: 187, at most 85% amino acid identity with SEQ ID NO: 187, at most 90% amino acid identity with SEQ ID NO: 187 or at most 95% amino acid identity with SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 187. In other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 187. In still other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 187. In other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 187. In still other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 187. In yet other aspects of this embodiment, a chromogranin C peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 187.

Another example of a binding domain disclosed in the present specification is, e.g., a tachykinin peptide, such as, e.g., a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. Thus, in an embodiment, a binding domain comprises a tachykinin peptide. In an embodiment, a binding domain is derived from a tachykinin peptide.

In aspects of this embodiment, a tachykinin peptide binding domain comprises a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. In aspects of this embodiment, a tachykinin peptide binding domain is derived from a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. In other aspects of this embodiment, a tachykinin peptide binding domain comprises SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 or SEQ ID NO: 199.

In other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 75% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 80% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 85% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 90% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199 or at least 95% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at most 75% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at most 80% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at most 85% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at most 90% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199 or at most 95% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199.

In other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In still other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199.

In other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In still other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of this embodiment, a tachykinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199.

Another example of a binding domain disclosed in the present specification is, e.g., a cholecystokinin peptide, such as, e.g., a cholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, a cholecystokinin 12 or a cholecystokinin 8. Thus, in an embodiment, a binding domain comprises a cholecystokinin peptide. In another embodiment, a binding domain is derived from a cholecystokinin peptide.

In aspects of this embodiment, a cholecystokinin peptide binding domain comprises a cholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, a cholecystokinin 12 or a cholecystokinin 8. In other aspects of this embodiment, a cholecystokinin peptide comprising a binding domain is SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises amino acids 20-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises amino acids 26-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still further other aspects of this embodiment, a cholecystokinin peptide binding domain comprises amino acids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210 or SEQ ID NO: 214. In yet further aspects of this embodiment, a cholecystokinin peptide binding domain comprises amino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at least 75% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at least 80% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at least 85% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at least 90% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215 or at least 95% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 75% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 80% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 85% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 90% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215 or at most 95% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.

In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at least 75% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at least 80% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at least 85% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at least 90% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200 or at least 95% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most 75% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most 80% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most 85% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most 90% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200 or at most 95% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200.

In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200.

In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at least 75% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at least 80% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at least 85% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at least 90% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200 or at least 95% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most 75% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most 80% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most 85% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most 90% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200 or at most 95% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid substitutions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid substitutions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four non-contiguous amino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four non-contiguous amino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid substitutions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid substitutions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In still other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three or four contiguous amino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, a cholecystokinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three or four contiguous amino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200.

Another example of a binding domain disclosed in the present specification is, e.g., a Neuropeptide Y related peptide, such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide (PIP). Thus, in an embodiment, a binding domain comprises a Neuropeptide Y related peptide. In another embodiment, a binding domain is derived from a Neuropeptide Y related peptide.

In aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide (PIP). In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at least 75% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at least 80% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at least 85% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at least 90% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220 or at least 95% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at most 75% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at most 80% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at most 85% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at most 90% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220 or at most 95% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In still other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In still other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Y related peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

Another example of a binding domain disclosed in the present specification is, e.g., a corticotropin-releasing hormone, a thyrotropin-releasing hormone, somatostatin, a leukemia inhibitor factor (LIF) or an interleukin-1 (IL1). Thus, in an embodiment, a binding domain comprises a corticotropin-releasing hormone. In an embodiment, a binding domain is derived from a corticotropin-releasing hormone. In another embodiment, a binding domain comprises a thyrotropin-releasing hormone. In another embodiment, a binding domain is derived from a thyrotropin-releasing hormone. In another embodiment, a binding domain comprises a somatostatin. In another embodiment, a binding domain is derived from a somatostatin. In another embodiment, a binding domain comprises a LIF. In another embodiment, a binding domain is derived from a LIF. In another embodiment, a binding domain comprises an IL1. In another embodiment, a binding domain is derived from an IL1. In aspects of this embodiment, a binding domain comprises SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

In other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, or SEQ ID NO: 226, at least 75% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at least 80% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at least 85% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at least 90% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226 or at least 95% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at most 75% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at most 80% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at most 85% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at most 90% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226 or at most 95% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

In other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In still other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

In other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In still other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yet other aspects of this embodiment, a binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

Another example of a binding domain disclosed in the present specification is a kinin peptide, such as, e.g., a bradykinin, a kallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin. Thus, in an embodiment, a binding domain comprises a kinin peptide. In another embodiment, a binding domain is derived from a kinin peptide. In aspects of this embodiment, a kinin peptide binding domain comprises a bradykinin, a kallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin. In other aspects of this embodiment, a kinin peptide binding domain comprises SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230.

In other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at least 75% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at least 80% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at least 85% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at least 90% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230 or at least 95% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 75% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 80% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 85% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 90% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230 or at most 95% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230.

In other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In still other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230.

In other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In still other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230.

Another example of a binding domain disclosed in the present specification is a PAR peptide, such as, e.g., a PAR1 peptide, a PAR2 peptide, a PAR3 peptide and a PAR4 peptide. Thus, in an embodiment, a binding domain comprises a PAR peptide. In another embodiment, a binding domain is derived from a PAR peptide. In aspects of this embodiment, a PAR peptide binding domain comprises a PAR1 peptide, a PAR2 peptide, a PAR3 peptide or a PAR4 peptide. In other aspects of this embodiment, a PAR peptide binding domain comprises amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at least 75% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at least 80% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at least 85% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at least 90% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234 or at least 95% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most 70% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at most 75% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at most 80% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at most 85% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234, at most 90% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234 or at most 95% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid substitutions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid substitutions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid deletions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In still other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five non-contiguous amino acid additions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five non-contiguous amino acid additions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In still other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at least one, two, three, four or five contiguous amino acid additions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptide binding domain comprises a polypeptide having, e.g., at most one, two, three, four or five contiguous amino acid additions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

Another example of a binding domain, includes, without limitation, a translocator, such as, e.g., a protein translocation domain (PTD), like, a herpes simplex virus type 1 VP22 protein translocating sequence, a SV-40 virus large T translocating sequence, a TAT translocating sequence, an adenovirus translocating sequence, a synthetic integrin binding domain translocating sequence, a Kaposi fibroblast growth factor membrane translocating sequence, a nuclear localization signal, a Transportan translocating sequence, a ciliary neurotrophic factor translocating sequence, a caveolin, an interleukin 1-β translocating sequence, a thioredoxin translocating sequence, a fibroblast growth factor-1 translocating sequence, a fibroblast growth factor-2 translocating sequence, an integrin β1 translocating sequence, an integrin β3 translocating sequence, a lactoferrin translocating sequence, a homeodomain translocating sequence, like, a penetratin translocating sequence, an Engrailed-1 translocating sequence, an Engrailed-2 translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4 translocating sequence, a Hoxc-8 translocating sequence.

Without wishing to limit the invention to any theory or mechanism of operation, it is believed that the translocator comprises a protein translocation domain (PTD). Further, it is believed that the PTD is primarily responsible for the translocation of the neurotoxin across a cell membrane. PTDs are amino acid sequence domains that have been shown to cross biological membranes efficiently and independently of transporters or specific receptors. See e.g., Morris M. C. et al., NATURE BIOTECHNOLOGY, 19:1173-1176 (December 2001), the disclosure of which is herein incorporated by reference in its entirety.

For example, herpes simplex virus type 1 viral protein 22 (HSV-1 VP 22) protein is a transcription factor that concentrates in the nucleus and binds chromatin. It has been shown that HSV-1 VP 22 comprises a translocating sequence that mediates trafficking across the membrane via non-classical endocytosis and can enter cells regardless of GAP junctions and physical contacts. If HSV-1 VP 22 is expressed in a small population of cells in culture, it will reach 100% of the cells in that culture. Fusion proteins with HSV-1 VP 22 and for example p53, GFP, thymidine kinase, β-galactosidase and others have been generated. It has been demonstrated that the fusion proteins are taken up by several kinds of cells including terminally differentiated cells suggesting that mitosis is not a requirement for efficient entry. In addition, HSV-1 VP 22-GFP fusion showed that the protein can shuttle in and out of the cells and enter cells that were not exposed to HSV-1 VP 22.

As another example, the trans-activator gene product (TAT) from the lentivirus HIV-1 was one of the earliest described cell-permeating proteins comprising a translocating sequence. A receptor-mediated event appears not to be required for HIV TAT to pass into a neighboring cell; thus for selective cell targeting, a PTD is preferably used in conjunction with a more target cell-selective binding domain in the multivalent Clostridial toxin derivative of the present invention. It is now known that many other lentiviruses, encodes a potent TAT protein. Embury et al., DIABETES 50:1706-1713, 2001 discuss the use the TAT PTD to transduce anti-apoptotic proteins into pancreatic islet cells. The PTD of TAT is a small peptide comprising amino acids 47-57 of the TAT protein; the PTD sequence comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO 41) or at least the nonapeptide comprising TAT amino acids 49-57. Fusion proteins comprising this peptide can transit the plasma membrane in vitro and in vivo. E.g., Schwartz, J. J. et al., Peptide-mediated cellular delivery, Curr. Opin. Mol. Therapeutics. 2000, 2:162-7. The disclosures of these references are incorporated in their entirety by reference herein.

As another example, a translocating sequence may be derived from a homeoprotein. Homeoproteins are helix turn helix proteins that contain a 60 amino acid DNA-binding domain, the homeodomain. a translocating sequence may be derived from the homeodomain. One kind of translocating sequence may be derived from the family of Drosophila homeoproteins. Drosophila homeoproteins are involved in developmental processes and are able to translocate across neuronal membranes. The third helix of the homeodomain of just 16 amino acids, known as penetratin, is able to translocate molecules into live cells. When added to several cell types in culture, 100% of the cells were able to uptake the peptide. Internalization occurs both at 37° C. and 4° C., and thus appears to be neither receptor-mediated nor energy-dependent. Several penetrating peptides, including those of the penetratin family, have been developed and used to internalize cargo molecules into the cytoplasm and nucleus of several cell types in vivo and in vitro. The results suggest that the entry of penetratin peptides is mediated by tryptophan, phenylalanine, and/or glutamine residues. In addition, the retroinverse and all D-amino acid forms are also translocated efficiently using the penetratins, and non-α-helical structures are also internalized. See Prochiantz, A., Messenger proteins:homeoproteins, TAT and others, Curr Opin Cell Biol 2000, 12:400-6; and Schwartz, J J et al., Peptide-mediated cellular delivery, Curr Opin Mol Therapeutics 2000, 2:162-7. The disclosures of these references are incorporated in their entirety by reference herein.

Furthermore, PTD-transported proteins and peptides, such as fusion peptides, have been shown to generally retain their biological properties and functions once inside the cells. Further, the TAT is able to carry a variety of cargo molecules including nucleic acid molecules (DNA and RNA), and therapeutic drugs. The capability of this sequence to promote internalization of the peptide (and any cargo peptide) appears to be related to the strongly positive charge of the PTD sequence, and not to be inhibited at 4° C. or in the presence of endocytosis inhibitors. Thus, the PTD appears not to be dependent upon active transport or receptor mediated endocytosis for this activity. The PTD sequence is able to mediate the transduction of its cargo in a concentration dependent and receptor-, transporter-, and endocytosis-independent manner to 100% of the target cells in a given tissue or culture. Of special interest are the studies demonstrating that the PTD of TAT is able to deliver proteins in vivo to several different tissues when injected into animals.

A PTD may comprise a synthetic translocating sequence, such as, e.g., the synthetic translocating sequence disclosed in, e.g., WO 99/29721 and Ho, A. et al., Synthetic PTDs: enhanced transduction potential in vitro and in vivo, CANCER RES 2001, 61, 474-7. In addition, it has been demonstrated that a 9-mer of L-Arginine is 20 fold more efficient than the TAT-PTD at cellular uptake, and when a D-arginine oligomer was used the rate enhancement was >100 fold. See Wender, P A et al., The Design, Synthesis, And Evaluation Of Molecules That Enable Or Enhance Cellular Uptake: Peptoid Molecular Transporters, PROC. NATL. ACAD. SCI. USA 2000, 97:13003-13008. These data suggested that the guanidinium groups of TAT-PTD play a greater role than charge or backbone structure in mediating cellular uptake. Thus, a peptoid analogue containing a six-methylene spacer between the guanidine head group and backbone was synthesized. This peptoid exhibited enhanced cellular uptake when compared to TAT-PTD and even to the D-Arg peptide.

In addition to the proteins and peptides discussed above, other peptide-mediated delivery systems have been described and are well-known to those of skill in the art. These PTD-containing proteins and peptides include: MPG, SCWKn, (LARL)n, HA2, RGD, AlkCWK₁₈, DiCWK₁₈, DipaLytic, K₁₆RGD, Plae and Kplae. See Schwartz, J J et al., Peptide-mediated cellular delivery, Curr Opin Mol Therapeutics 2000, 2:162-7. The disclosure of which is incorporated in its entirety by reference herein. In some embodiments, these proteins and peptides may be used as translocators in accordance with the present invention.

A number of PTDs have now been characterized and are well-known in the art. The amino acid sequences of exemplary translocators are shown in Table 8.

TABLE 8 Translocator Peptides Useful as Binding Domains PTD Translocating Sequence SEQ ID NO: SV-40 virus large T CGGGPKKKRKVGG 235 TAT YGRKKRRQRRR 236 Adenovirus CGGFSTSLRARKA 237 Integrin binding domain CKKKKKKGGRGDMFG 238 Kaposi FGF membrane AAVALLPAVLLALLAP 239 translocating sequence Nuclear Localization Signal TPPKKKRKVEDP 240 Transportan GWTLNSAGYLLGKINLKALAALAKKIL 241 HSV-1 VP 22 DAATATRGRSAASRPTERPRAPARSASRPRRPVE 242 Penetratin 43-58 RQIKIWFQNRRMKWKK 243 Penetratin 58-43 KKWKMRRNQFWIKIQR 244 Penetratin 43-58 RQIKIWFQNRRMKWKK 245 Penetratin Pro50 RQIKIWFPNRRMKWKK 246 Penetratin 3Pro RQPKIWFPNRRMPWKK 247 Penetratin Met-Arg RQIKIWFQNMRRKWKK 248 Penetratin 7Arg RQIRIWFQNRRMRWRR 249 Penetratin W/R RRWRRWWRRWWRRWRR 250 Penetratin-1 RQIKIFFQNRRMKFKK 251 Penetratin-2 TERQIKIWFQNRRMK 252 Penetratin-3 KIWFQNRRMKWKKEN 253

As used herein, a “translocator” of the present invention comprises a polypeptide or a peptidomimetic that facilitates the transport of a molecule across a cell membrane. It is envisioned that both naturally occurring translocators as well as non-naturally occurring translocators can be used as a binding domain. A translocator includes, without limitation, naturally occurring translocator variants, such as, e.g., translocator isoforms and translocator subtypes; non-naturally occurring translocator variants, such as, e.g., conservative translocator variants, non-conservative translocator variants, translocator chimerics, active translocator fragments thereof, or any combination thereof.

As used herein, the term “translocator variant,” whether naturally-occurring or non-naturally-occurring, means a translocator that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Table 8) and can be described in percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all translocator variants disclosed in the present specification are capable of executing the cell binding step of the intoxication process.

It is recognized by those of skill in the art that there can be naturally occurring translocator variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, HSV-1 VP22 protein variants, TAT variants, and penetratin variants. As used herein, the term “naturally occurring translocator variant” means any translocator produced by a naturally-occurring process, including, without limitation, translocator isoforms produced from alternatively-spliced transcripts, translocator isoforms produced by spontaneous mutation and translocator subtypes. A naturally occurring translocator variant can function in substantially the same manner as the reference translocator on which the naturally occurring translocator variant is based, and can be substituted for the reference translocator in any aspect of the present invention. A naturally occurring translocator variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, seven or more amino acids, eight or more amino acids, nine or more amino acids, or ten or more amino acids from the reference translocator on which the naturally occurring translocator variant is based. A naturally occurring translocator variant can also substitute at least 2 contiguous amino acids, at least 3 contiguous amino acids, at least 4 contiguous amino acids, at least 5 contiguous amino acids, at least 6 contiguous amino acids, at least 7 contiguous amino acids, at least 8 contiguous amino acids, at least 9 contiguous amino acids, or at least 10 contiguous amino acids from the reference translocator on which the naturally occurring translocator variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference translocator on which the naturally occurring translocator variant is based.

A non-limiting examples of a naturally occurring translocator variant is a translocator isoform such as, e.g., a HSV-1 VP22 protein isoform, a SV-40 virus large T isoform, a TAT isoform, an adenovirus isoform, a synthetic integrin binding domain isoform, a Kaposi fibroblast growth factor membrane isoform, a nuclear localization signal, a Transportan isoform, a ciliary neurotrophic factor isoform, a caveolin, an interleukin 1-β isoform, a thioredoxin isoform, a fibroblast growth factor-1 isoform, a fibroblast growth factor-2 isoform, an integrin β1 isoform, an integrin β3 isoform, a lactoferrin isoform, a homeodomain isoform, like, a penetratin isoform, an Engrailed-1 isoform, an Engrailed-2 isoform, a Hoxa-5 isoform, a Hoxb-4 isoform, a Hoxc-8 isoform. A translocator isoform can function in substantially the same manner as the reference translocator on which the translocator isoform is based, and can be substituted for the reference translocator in any aspect of the present invention.

Another non-limiting examples of a naturally occurring translocator variant is a translocator subtype such as, e.g., a HSV-1 VP22 protein subtype, a SV-40 virus large T subtype, a TAT subtype, an adenovirus subtype, a synthetic integrin binding domain subtype, a Kaposi fibroblast growth factor membrane subtype, a nuclear localization signal, a Transportan subtype, a ciliary neurotrophic factor subtype, a caveolin, an interleukin 1-β subtype, a thioredoxin subtype, a fibroblast growth factor-1 subtype, a fibroblast growth factor-2 subtype, an integrin β1 subtype, an integrin β3 subtype, a lactoferrin subtype, a homeodomain subtype, like, a penetratin subtype, an Engrailed-1 subtype, an Engrailed-2 subtype, a Hoxa-5 subtype, a Hoxb-4 subtype, a Hoxc-8 subtype. A translocator subtype can function in substantially the same manner as the reference translocator on which the translocator subtype is based, and can be substituted for the reference translocator in any aspect of the present invention.

As used herein, the term “non-naturally occurring translocator variant” means any translocator produced with the aid of human manipulation, including, without limitation, translocators produced by genetic engineering using random mutagenesis or rational design and translocators produced by chemical synthesis. Non-limiting examples of non-naturally occurring translocator variants include, e.g., conservative translocator variants, non-conservative translocator variants, translocator chimeric variants and active translocator fragments.

As used herein, the term “conservative translocator variant” means a translocator that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference translocator sequence (see Table 8). Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative translocator variant can function in substantially the same manner as the reference translocator on which the conservative translocator variant is based, and can be substituted for the reference translocator in any aspect of the present invention. A conservative translocator variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, seven or more amino acids, eight or more amino acids, nine or more amino acids, or ten or more amino acids from the reference translocator on which the conservative translocator variant is based. A conservative translocator variant can also substitute at least 2 contiguous amino acids, at least 3 contiguous amino acids, at least 4 contiguous amino acids, at least 5 contiguous amino acids, at least 6 contiguous amino acids, at least 7 contiguous amino acids, at least 8 contiguous amino acids, at least 9 contiguous amino acids, or at least 10 contiguous amino acids from the reference translocator on which the conservative translocator variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference translocator on which the conservative translocator variant is based. Non-limiting examples of a conservative translocator variant include, e.g., a conservative HSV-1 VP22 protein variant, a conservative SV-40 virus large T variant, a conservative TAT variant, an adenovirus variant, a conservative synthetic integrin binding domain variant, a conservative Kaposi fibroblast growth factor membrane variant, a conservative nuclear localization signal, a conservative Transportan variant, a conservative ciliary neurotrophic factor variant, a conservative caveolin, an interleukin 1-β variant, a conservative thioredoxin variant, a conservative fibroblast growth factor-1 variant, a conservative fibroblast growth factor-2 variant, an integrin β1 variant, an integrin β3 variant, a conservative lactoferrin variant, a conservative homeodomain variant, like, a conservative penetratin variant, an Engrailed-1 variant, an Engrailed-2 variant, a conservative Hoxa-5 variant, a conservative Hoxb-4 variant, a conservative Hoxc-8 variant.

As used herein, the term “non-conservative translocator variant” means a translocator in which 1) at least one amino acid is deleted from the reference translocator on which the non-conservative translocator variant is based; 2) at least one amino acid added to the reference translocator on which the non-conservative translocator is based; or 3) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to that of the original amino acid from the reference translocator sequence (see Table 8). A non-conservative translocator variant can function in substantially the same manner as the reference translocator on which the non-conservative translocator variant is based, and can be substituted for the reference translocator in any aspect of the present invention. A non-conservative translocator variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference translocator on which the non-conservative translocator variant is based. A non-conservative translocator variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference translocator on which the non-conservative translocator variant is based. A non-conservative translocator variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, seven or more amino acids, eight or more amino acids, nine or more amino acids, or ten or more amino acids from the reference translocator on which the non-conservative translocator variant is based. A non-conservative translocator variant can also substitute at least 2 contiguous amino acids, at least 3 contiguous amino acids, at least 4 contiguous amino acids, at least 5 contiguous amino acids, at least 6 contiguous amino acids, at least 7 contiguous amino acids, at least 8 contiguous amino acids, at least 9 contiguous amino acids, or at least 10 contiguous amino acids from the reference translocator on which the non-conservative translocator variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference translocator on which the non-conservative translocator variant is based. Non-limiting examples of a non-conservative translocator variant include, e.g., a non-conservative HSV-1 VP22 protein variant, a non-conservative SV-40 virus large T variant, a non-conservative TAT variant, an adenovirus variant, a non-conservative synthetic integrin binding domain variant, a non-conservative Kaposi fibroblast growth factor membrane variant, a non-conservative nuclear localization signal, a non-conservative Transportan variant, a non-conservative ciliary neurotrophic factor variant, a non-conservative caveolin, an interleukin 1-β variant, a non-conservative thioredoxin variant, a non-conservative fibroblast growth factor-1 variant, a non-conservative fibroblast growth factor-2 variant, an integrin β1 variant, an integrin β3 variant, a non-conservative lactoferrin variant, a non-conservative homeodomain variant, like, a non-conservative penetratin variant, an Engrailed-1 variant, an Engrailed-2 variant, a non-conservative Hoxa-5 variant, a non-conservative Hoxb-4 variant, a non-conservative Hoxc-8 variant.

As used herein, the term “translocator chimeric” means a polypeptide comprising at least a portion of a translocator and at least a portion of at least one other polypeptide to form an enhanced targeting domain with at least one property different from the reference translocator (see Table 8), with the proviso that this translocator chimeric can facilitate the transport of a molecule across a cell membrane.

As used herein, the term “active translocator fragment” means any of a variety of translocator fragments comprising the enhanced targeting domain can be useful in aspects of the present invention with the proviso that these NAP fragments can facilitate the transport of a molecule across a cell membrane.

In some embodiments, the translocator may function independently of cell surface transporters or specific receptors, and therefore non-specifically. In this embodiment the multivalent Clostridial toxin will also comprise at least one target cell selective ligand. In some embodiments, one or more binding domain of the multivalent Clostridial toxin may comprise a translocator. In other embodiments, a translocator may comprise a peptide or peptidomimetic selective for a cell surface transporter, feature, or receptor; in such an embodiment the multivalent Clostridial toxin may comprise two or more identical binding domains, or may comprise at least one target cell selective domain and at least one target cell non-selective binding domain, so long as the number of binding domains totals at least two.

In an embodiment, a binding domain comprises a translocator. In aspects of this embodiment, a translocator comprises a PTD.

In another embodiment, a binding domain comprises a translocating sequence comprises a SV-40 virus large T translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a SV-40 virus large T translocating sequence. In an aspect of this embodiment, a binding domain comprises a SV-40 virus large T translocating sequence comprises SEQ ID NO: 235. In another aspect of this embodiment, a binding domain comprises a SV-40 virus large T translocating sequence derived from SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 235, at least 75% amino acid identity with SEQ ID NO: 235, at least 80% amino acid identity with SEQ ID NO: 235, at least 85% amino acid identity with SEQ ID NO: 235, at least 90% amino acid identity with SEQ ID NO: 235 or at least 95% amino acid identity with SEQ ID NO: 235. In yet other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 235, at most 75% amino acid identity with SEQ ID NO: 235, at most 80% amino acid identity with SEQ ID NO: 235, at most 85% amino acid identity with SEQ ID NO: 235, at most 90% amino acid identity with SEQ ID NO: 235 or at most 95% amino acid identity with SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 235. In yet other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 235. In still other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 235. In yet other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 235. In still other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 235. In other aspects of this embodiment, a SV-40 virus large T translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 235.

In another embodiment, a binding domain comprises a translocating sequence comprises a TAT translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a TAT translocating sequence. In an aspect of this embodiment, a binding domain comprises a TAT translocating sequence comprises SEQ ID NO: 236. In another aspect of this embodiment, a binding domain comprises a TAT translocating sequence derived from SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 236, at least 75% amino acid identity with SEQ ID NO: 236, at least 80% amino acid identity with SEQ ID NO: 236, at least 85% amino acid identity with SEQ ID NO: 236, at least 90% amino acid identity with SEQ ID NO: 236 or at least 95% amino acid identity with SEQ ID NO: 236. In yet other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 236, at most 75% amino acid identity with SEQ ID NO: 236, at most 80% amino acid identity with SEQ ID NO: 236, at most 85% amino acid identity with SEQ ID NO: 236, at most 90% amino acid identity with SEQ ID NO: 236 or at most 95% amino acid identity with SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 236. In yet other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 236. In still other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 236. In yet other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 236. In still other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 236. In other aspects of this embodiment, a TAT translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 236.

In another embodiment, a binding domain comprises a translocating sequence comprises an adenovirus translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from an adenovirus translocating sequence. In an aspect of this embodiment, a binding domain comprises an adenovirus translocating sequence comprises SEQ ID NO: 237. In another aspect of this embodiment, a binding domain comprises an adenovirus translocating sequence derived from SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 237, at least 75% amino acid identity with SEQ ID NO: 237, at least 80% amino acid identity with SEQ ID NO: 237, at least 85% amino acid identity with SEQ ID NO: 237, at least 90% amino acid identity with SEQ ID NO: 237 or at least 95% amino acid identity with SEQ ID NO: 237. In yet other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 237, at most 75% amino acid identity with SEQ ID NO: 237, at most 80% amino acid identity with SEQ ID NO: 237, at most 85% amino acid identity with SEQ ID NO: 237, at most 90% amino acid identity with SEQ ID NO: 237 or at most 95% amino acid identity with SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 237. In yet other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 237. In still other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 237. In yet other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 237. In still other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 237. In other aspects of this embodiment, an adenovirus translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 237.

In another embodiment, a binding domain comprises a translocating sequence comprises an integrin binding domain translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from an integrin binding domain translocating sequence. In an aspect of this embodiment, a binding domain comprises an integrin binding domain translocating sequence comprises SEQ ID NO: 238. In another aspect of this embodiment, a binding domain comprises an integrin binding domain translocating sequence derived from SEQ ID NO: 238.

In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 238, at least 75% amino acid identity with SEQ ID NO: 238, at least 80% amino acid identity with SEQ ID NO: 238, at least 85% amino acid identity with SEQ ID NO: 238, at least 90% amino acid identity with SEQ ID NO: 238 or at least 95% amino acid identity with SEQ ID NO: 238. In yet other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 238, at most 75% amino acid identity with SEQ ID NO: 238, at most 80% amino acid identity with SEQ ID NO: 238, at most 85% amino acid identity with SEQ ID NO: 238, at most 90% amino acid identity with SEQ ID NO: 238 or at most 95% amino acid identity with SEQ ID NO: 238.

In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 238. In yet other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 238. In still other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 238.

In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 238. In yet other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 238. In still other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 238. In other aspects of this embodiment, an integrin binding domain translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 238.

In another embodiment, a binding domain comprises a translocating sequence comprises a Kaposi FGF membrane translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a Kaposi FGF membrane translocating sequence. In an aspect of this embodiment, a binding domain comprises a Kaposi FGF membrane translocating sequence comprises SEQ ID NO: 239. In another aspect of this embodiment, a binding domain comprises a Kaposi FGF membrane translocating sequence derived from SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 239, at least 75% amino acid identity with SEQ ID NO: 239, at least 80% amino acid identity with SEQ ID NO: 239, at least 85% amino acid identity with SEQ ID NO: 239, at least 90% amino acid identity with SEQ ID NO: 239 or at least 95% amino acid identity with SEQ ID NO: 239. In yet other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 239, at most 75% amino acid identity with SEQ ID NO: 239, at most 80% amino acid identity with SEQ ID NO: 239, at most 85% amino acid identity with SEQ ID NO: 239, at most 90% amino acid identity with SEQ ID NO: 239 or at most 95% amino acid identity with SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 239. In yet other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 239. In still other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 239. In yet other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 239. In still other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 239. In other aspects of this embodiment, a Kaposi FGF membrane translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 239.

In another embodiment, a binding domain comprises a translocating sequence comprises a Nuclear Localization Signal translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a Nuclear Localization Signal translocating sequence. In an aspect of this embodiment, a binding domain comprises a Nuclear Localization Signal translocating sequence comprises SEQ ID NO: 240. In another aspect of this embodiment, a binding domain comprises a Nuclear Localization Signal translocating sequence derived from SEQ ID NO: 240.

In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 240, at least 75% amino acid identity with SEQ ID NO: 240, at least 80% amino acid identity with SEQ ID NO: 240, at least 85% amino acid identity with SEQ ID NO: 240, at least 90% amino acid identity with SEQ ID NO: 240 or at least 95% amino acid identity with SEQ ID NO: 240. In yet other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 240, at most 75% amino acid identity with SEQ ID NO: 240, at most 80% amino acid identity with SEQ ID NO: 240, at most 85% amino acid identity with SEQ ID NO: 240, at most 90% amino acid identity with SEQ ID NO: 240 or at most 95% amino acid identity with SEQ ID NO: 240.

In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 240. In yet other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 240. In still other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 240.

In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 240. In yet other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 240. In still other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 240. In other aspects of this embodiment, a Nuclear Localization Signal translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 240.

In another embodiment, a binding domain comprises a translocating sequence comprises a Transportan translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a Transportan translocating sequence. In an aspect of this embodiment, a binding domain comprises a Transportan translocating sequence comprises SEQ ID NO: 241. In another aspect of this embodiment, a binding domain comprises a Transportan translocating sequence derived from SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 241, at least 75% amino acid identity with SEQ ID NO: 241, at least 80% amino acid identity with SEQ ID NO: 241, at least 85% amino acid identity with SEQ ID NO: 241, at least 90% amino acid identity with SEQ ID NO: 241 or at least 95% amino acid identity with SEQ ID NO: 241. In yet other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 241, at most 75% amino acid identity with SEQ ID NO: 241, at most 80% amino acid identity with SEQ ID NO: 241, at most 85% amino acid identity with SEQ ID NO: 241, at most 90% amino acid identity with SEQ ID NO: 241 or at most 95% amino acid identity with SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 241. In yet other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 241. In still other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 241. In yet other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 241. In still other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 241. In other aspects of this embodiment, a Transportan translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 241.

In another embodiment, a binding domain comprises a translocating sequence comprises a HSV-1 VP 22 translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a HSV-1 VP 22 translocating sequence. In an aspect of this embodiment, a binding domain comprises a HSV-1 VP 22 translocating sequence comprises SEQ ID NO: 242. In another aspect of this embodiment, a binding domain comprises a HSV-1 VP 22 translocating sequence derived from SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 242, at least 75% amino acid identity with SEQ ID NO: 242, at least 80% amino acid identity with SEQ ID NO: 242, at least 85% amino acid identity with SEQ ID NO: 242, at least 90% amino acid identity with SEQ ID NO: 242 or at least 95% amino acid identity with SEQ ID NO: 242. In yet other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 242, at most 75% amino acid identity with SEQ ID NO: 242, at most 80% amino acid identity with SEQ ID NO: 242, at most 85% amino acid identity with SEQ ID NO: 242, at most 90% amino acid identity with SEQ ID NO: 242 or at most 95% amino acid identity with SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 242. In yet other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 242. In still other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 242. In yet other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 242. In still other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 242. In other aspects of this embodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 242.

In another embodiment, a binding domain comprises a translocating sequence comprises a Penetratin translocating sequence. In another embodiment, a binding domain comprises a translocating sequence derived from a Penetratin translocating sequence. In an aspect of this embodiment, a binding domain comprises a Penetratin translocating sequence comprises SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253. In another aspect of this embodiment, a binding domain comprises a Penetratin translocating sequence derived from SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.

In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 243, at least 75% amino acid identity with SEQ ID NO: 243, at least 80% amino acid identity with SEQ ID NO: 243, at least 85% amino acid identity with SEQ ID NO: 243, at least 90% amino acid identity with SEQ ID NO: 243 or at least 95% amino acid identity with SEQ ID NO: 243. In yet other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most 70% amino acid identity with SEQ ID NO: 243, at most 75% amino acid identity with SEQ ID NO: 243, at most 80% amino acid identity with SEQ ID NO: 243, at most 85% amino acid identity with SEQ ID NO: 243, at most 90% amino acid identity with SEQ ID NO: 243 or at most 95% amino acid identity with SEQ ID NO: 243.

In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid substitutions relative to SEQ ID NO: 243. In yet other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 243. In still other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 non-contiguous amino acid additions relative to SEQ ID NO: 243.

In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO: 243. In yet other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 243. In still other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 243. In other aspects of this embodiment, a Penetratin translocating sequence comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 243.

Another example of a binding domain, includes, without limitation, an antibody to a coated pit protein, such as, e.g., a clatherin antibody and an Adaptor Protein-2 (adaptin) antibody; an antibody to a caveolae-associated protein, such as, e.g., a caveolin-1 antibody and a GPI-linked receptor protein antibody.

One major route of receptor-mediated endocytosis is by means of endocytotic coated pits. These specialized sites on the plasma membrane of eukaryotic cells are responsible for internalization of many cell surface receptors, usually after ligand binding. The coated pits serve in part to concentrate the receptors for internalization within far fewer endosomes than would be the case if each receptor were individually internalized. Coated pits consist largely of two major proteins: clatherin and the adaptor protein (adaptin) Adaptor Protein-2 (AP-2). Assembly of the coated pit is thought to be initiated by the binding of AP-2 molecules to a receptor or ligand-docking site in the membrane. The receptor:AP-2 complex then recruits the structural protein clatherin.

Sorting signals present in many membrane proteins, such as cell surface receptors, provide for recognition and binding of AP-2 to these proteins as the initial step of coated pit formation. One common sorting signal is the amino acid sequence YXXΦ (wherein Φ is an amino acid with a bulky hydrophobic side chain, such as leucine, phenylalanine, methionine and valine), which provides a signal for rapid internalization. This signal is recognized by and binds to the μ2 subunit of AP-2, and mediates rapid internalization by mammalian cells. Preferably, the signal is present ion such a way as to be displayed on the cytosolic side of the membrane. See e.g., Bonifacino et al., J. Cell Biol. 145: 923 (May 31, 1999).

At least one binding domain of a multivalent Clostridial toxin of the present invention may comprise at least one sorting signal for an adaptin, preferably AP-2. Preferably, the binding site comprising this sorting signal is present in addition to another distinct binding domain which will directly bind a cell surface receptor.

In certain embodiments, a multivalent Clostridial toxin of the present invention comprising two or more pancreatic acinar cell targeting moieties may be administered to treat pancreatitis; the use of CCK forms and derivatives capable of selective binding to CCK receptors of pancreatic acinar cells is disclosed in U.S. Pat. No. 6,843,998. In one preferred embodiment of this aspect of the present invention, two or more binding moieties of the present invention are independently a CCK or CCK derivative.

In another embodiment of the invention for treatment of, e.g., acute or chronic pancreatitis, at least one binding moiety of the multivalent Clostridial toxin is a CCK or CCK derivative, and at least one additional binding region comprises a region of a glycosyl phosphatylinositol (GPI)-linked membrane protein that directly or indirectly binds caveolin-1 binding. Alternatively, the additional binding region may comprise a ligand capable of binding a GPI-linked membrane protein, particularly a membrane associated cell surface receptor, as the CCK receptor, like the TNF and other known receptors, is known to be concentrated and recruited for internalization by caveolae.

Caveolae are discrete membrane domains present in many cell types; they are not thought to be present in neural cells. Caveolae are comprised largely of detergent-insoluble lipids and lipid associated proteins, including a major protein, caveolin-1. Caveolae are also known to recruit and concentrate GPI-linked membrane proteins, a family of membrane proteins that includes cell surface receptors. Further, caveolae appear to participate in a non clatherin-associated form of endocytosis, and experiments have been reported in which gene transfer vectors have been directed to GPI-linked receptor proteins to facilitate gene transfer.

Thus, for example, the urokinase plasminogen activator receptor (uPAR) is a GPI-linked membrane protein that appears to facilitate endocytosis upon binding of its cognate ligand urokinase plasminigen activator (uPA). See e.g., Drapkin et al., J. CLIN. INVEST. 105: 589-596 (Mar. 1, 2000), incorporated by reference as part of this specification. uPA is a 55 KDa protein secreted by alveolar epithelial cells that cleaves plasminogen to plasmin and is known to degrade the extracellular matrix in alveoli. Targeting such cells, which also display the uPAR, with the multivalent Clostridial toxin of the present invention comprising, for example, two or more binding domains comprising a uPAR-binding region derived from, for example, uPA or an anti-uPAR antibody may be of therapeutic use is the treatment of respiratory and lung diseases such as e.g., cystic fibrosis, since the ligand-bound receptor is subject to endocytosis in a ligand dependent manner. Thus, for example, a patent may be administered a mist or dispersion comprising such multivalent Clostridial toxin as a delivery mechanism.

In certain embodiments the target cell may comprise a cell comprising a membrane protein that displays an antibody variable region at the cell surface. In such a case the multivalent Clostridial toxin comprises two or more binding domains, with at least one binding domain comprising an antigen able to selectively bind the antibody variable region. In a preferred embodiment, the multivalent Clostridial toxin may comprise more than one binding region comprising an antigen able to selectively bind to said antibody variable region.

Alternatively, the multivalent Clostridial toxin of the present invention (comprising more than one binding domain), may have at least one binding domain comprising an antigen-binding portion of an antibody H or L chain, wherein the antigen comprises a cell surface marker for a target cell. For example, and without limitation, the multivalent Clostridial toxin of the present invention may comprise at least one binding domain comprising an antibody variable region that selectively binds to an eosinophil cell surface marker (for example, the cell surface markers CD 44 and CD 69), thereby aiding in alleviating the symptoms of allergy. The nomenclature “CD” is derived from the use of monoclonal antibodies directed towards a given cell type, and stands for “Cluster of Differentiation”.

In another example, the multivalent Clostridial toxin derivative may be used to inhibit or decrease the rate of infection of T helper cells by the human immunodeficiency virus (HIV). T helper cells are commonly identified by virtue of their display of the cell surface marker CD4. The HIV virus uses the CD4 marker to gain entry into and thereby infect T helper cells, and appears to employ viral fusion proteins sharing remarkable similarity to the SNARE system to invade cells through a membrane fusion mechanism. See e.g., Duman & Forte, AM. J. PHYSIOL. CELL PHYSIOL. 285: C237-C249 (2003). A multivalent Clostridial toxin of the present invention comprising at least one, and preferably two or more, anti-CD4 antibody domain or similar CD4 “addressable” binding domain would permit the neurotoxin protease domain to deny the virus use of the SNARE proteins for entry and/or exit of the cell. Moreover, alteration of the toxin proteolytic domain to selectively recognize and cleave one or more of the HIV viral fusion proteins, using such techniques as directed evolution, site directed mutagenesis or other well known molecular biology methods, could provide a method of reducing the extent of, or eliminating, HIV infection.

In another aspect of the invention, a multivalent Clostridial toxin comprises, in part, a protease cleavage site. As used herein, the term “protease cleavage site” means a scissile bond together with adjacent or non-adjacent recognition elements, or both, sufficient for detectable proteolysis at the scissile bond by a protease under conditions suitable for protease activity. A protease cleavage site can be an endogenous protease cleavage site or an exogenous protease cleavage site. One function of the protease cleavage site may be to convert the single-chain polypeptide form of a Clostridial toxin into the di-chain form. Another role of a protease cleavage site may be change the conformational structure of a binding domain, thereby facilitating the binding domain's ability to bind to its cognate receptor, e.g., a binding domain may function only as a dimer, like the binding domains from the TGFβ superfamily; or exposing the amino terminal end of a binding domain, like the binding domains of opiod family members. Likewise, the location and kind of protease cleavage site may be critical because certain targeting domains require a free amino-terminal or carboxyl-terminal amino acid. For example, when a targeting domain is placed between two other domains, one criterion for selection of a protease cleavage site could be whether the protease that cleaves its site leaves a flush cut, exposing the free amino-terminal or carboxyl-terminal of the altered targeting domain necessary for selective binding of the targeting domain to its receptor. The selection and placement of a protease cleavage site is well known in the art.

It is envisioned that a multivalent Clostridial toxin may comprise one protease cleavage site. It is also envisioned that a multivalent Clostridial toxin may comprise a plurality of protease cleavage sites. Thus. in an embodiment, a multivalent Clostridial toxin may comprise one protease cleavage site. In another embodiment, a multivalent Clostridial toxin may comprise a plurality protease cleavage site. In aspects of this embodiment, a multivalent Clostridial toxin may comprise, e.g., at least one protease cleavage site, at least two protease cleavage sites, at least three protease cleavage sites, at least four protease cleavage sites, or at least five protease cleavage sites. In other aspects of this embodiment, a multivalent Clostridial toxin may comprise, e.g., at most one protease cleavage site, at most two protease cleavage sites, at most three protease cleavage sites, at most four protease cleavage sites, or at most five protease cleavage sites. In still other aspects of this embodiment, a multivalent Clostridial toxin may comprise, e.g., one protease cleavage site, two protease cleavage sites, three protease cleavage sites, four protease cleavage sites, or five protease cleavage sites.

It is envisioned that a multivalent Clostridial toxin can comprise an endogenous protease cleavage. As used herein, the term “endogenous protease cleavage site” is synonymous with “di-chain loop protease cleavage site,” “naturally occurring Clostridial toxin di-chain loop protease cleavage site” or “naturally occurring Clostridial toxin protease cleavage site” and means a naturally occurring Clostridial toxin protease cleavage site found within the di-chain loop region of a naturally occurring Clostridial toxin and includes, without limitation, naturally occurring Clostridial toxin di-chain loop protease cleavage site variants, such as, e.g., Clostridial toxin di-chain loop protease cleavage site isoforms and Clostridial toxin di-chain loop protease cleavage site subtypes. Non-limiting examples of an endogenous protease cleavage site, include, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/G di-chain loop protease cleavage site and a TeNT di-chain loop protease cleavage site.

As mentioned above, Clostridial toxins are translated as a single-chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulphide bond and noncovalent interactions. While the identity of the protease is currently unknown, the di-chain loop protease cleavage site for many Clostridial toxins has been proposed. In BoNTs, cleavage at K448-A449 converts the single polypeptide form of BoNT/A into the di-chain form; cleavage at K441-A442 converts the single polypeptide form of BoNT/B into the di-chain form; cleavage at K449-T450 converts the single polypeptide form of BoNT/C1 into the di-chain form; cleavage at R445-D446 converts the single polypeptide form of BoNT/D into the di-chain form; cleavage at R422-K423 converts the single polypeptide form of BoNT/E into the di-chain form; cleavage at K439-A440 converts the single polypeptide form of BoNT/F into the di-chain form; and cleavage at K446-S447 converts the single polypeptide form of BoNT/G into the di-chain form. Proteolytic cleavage of the single polypeptide form of TeNT at A457-S458 results in the di-chain form. Such a di-chain loop protease cleavage site is operably-linked in-frame to a multivalent Clostridial toxin as a fusion protein.

However, it should also be noted that additional cleavage sites within the di-chain loop also appear to be cleaved resulting in the generation of a small peptide fragment being lost. As a non-limiting example, BoNT/A single-chain polypeptide cleave ultimately results in the loss of a ten amino acid fragment within the di-chain loop. Thus, in BoNTs, cleavage at S441-L442 converts the single polypeptide form of BoNT/A into the di-chain form; cleavage at G444-1445 converts the single polypeptide form of BoNT/B into the di-chain form; cleavage at S445-L446 converts the single polypeptide form of BoNT/C1 into the di-chain form; cleavage at K442-N443 converts the single polypeptide form of BoNT/D into the di-chain form; cleavage at K419-G420 converts the single polypeptide form of BoNT/E into the di-chain form; cleavage at K423-S424 converts the single polypeptide form of BoNT/E into the di-chain form; cleavage at K436-G437 converts the single polypeptide form of BoNT/F into the di-chain form; cleavage at T444-G445 converts the single polypeptide form of BoNT/G into the di-chain form; and cleavage at E448-Q449 converts the single polypeptide form of BoNT/G into the di-chain form.

As used herein, the term “di-chain loop region” means the amino acid sequence of a Clostridial toxin containing a protease cleavage site used to convert the single-chain form of a Clostridial toxin into the di-chain form. Non-limiting examples of a Clostridial toxin di-chain loop region, include, a di-chain loop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; and a di-chain loop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8 (Table 9).

TABLE 9 Di-chain Loop Region of Clostridial Toxins Di-chain Loop Region SEQ Containing the Naturally- ID Light Chain occurring Protease Heavy Chain Toxin NO: Region Cleavage Site Region BoNT/A 1 NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLC IKVNNWDL BoNT/B 2  KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGIC IDVDNEDL BoNT/C1 3   PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDC RELLVKNTDL BoNT/D 4   PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTC IKVKNNRL BoNT/E 5    IITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSIC IEINNGEL BoNT/F 6    IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLC IRVNNSEL BoNT/G 7  KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQC IIVNNEDL TeNT 8  TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELC IKIKNEDL The amino acid sequence displayed are as follows: BoNT/A, residues 325-462 of SEQ ID No: 1; BoNT/B, residues 332-454 of SEQ ID No: 2; BoNT/C1, residues 334-463 of SEQ ID No: 3; BoNT/D, residues 334-458 of SEQ ID No: 4; BoNT/E, residues 311-434 of SEQ ID No: 5; BoNT/F, residues 328-453 of SEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No: 7; and TeNT, residues 334-474 of SEQ ID No: 8. An asterisks (*) indicates a peptide bond that is cleaved by a Clostridial toxin protease.

Thus, in an embodiment, a multivalent Clostridial toxin comprising a protease cleavage site comprises an endogenous protease cleavage site. In aspects of this embodiment, an endogenous protease cleavage site comprises, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/G di-chain loop protease cleavage site or a TeNT di-chain loop protease cleavage site.

In other aspects of this embodiment, an endogenous protease cleavage site comprises, e.g., a di-chain loop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; or a di-chain loop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8.

It is also envisioned that a multivalent Clostridial toxin can comprise an exogenous protease cleavage. As used herein, the term “exogenous protease cleavage site” is synonymous with a “non-naturally occurring Clostridial toxin protease cleavage site” and means a protease cleavage site that is not normally present in a di-chain loop region from a naturally occurring Clostridial toxin. Non-limiting examples of exogenous protease cleavage sites include, e.g., an enterokinase cleavage site (Table 10); a Thrombin cleavage site (Table 10); a Factor Xa cleavage site (Table 10); a human rhinovirus 3C protease cleavage site (Table 10); a tobacco etch virus (TEV) protease cleavage site (Table 10); a dipeptidyl aminopeptidase cleavage site; a small ubiquitin-like modifier (SUMO)/ubiquitin-like protein-1(ULP-1) protease cleavage site, such as, e.g., MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 271); and a Clostridial toxin substrate cleavage site.

As mentioned above, a Clostridial toxin is converted from a single polypeptide form into a di-chain molecule by proteolytic cleavage. While the naturally-occurring protease is currently not known, cleavage occurs within the di-chain loop region between the two cysteine residues that form the disulfide bridge (see Table 9). Replacement of an endogenous protease cleavage site with an exogenous protease cleavage site will enable cleavage of a multivalent Clostridial toxin disclosed in the present specification when expressed in an organism that does not produce the naturally-occurring protease used to cleave the di-chain loop region of a toxin. Similarly, an addition of an exogenous protease cleavage site in the di-chain loop region will also enable cleavage of a multivalent Clostridial toxin disclosed in the present specification when expressed in an organism that does not produce the naturally-occurring protease used to cleave the di-chain loop region of a toxin. Furthermore, cleavage at an exogenous protease cleavage site can facilitate a change in the conformational structure of a binding domain, thereby facilitating the binding domain's ability to bind to its cognate receptor, e.g., a binding domain may function only as a dimer, like the binding domains from the TGFβ superfamily; or exposing the amino terminal end of a binding domain, like the binding domains of opiod family members.

TABLE 10 Exogenous Protease Cleavage Sites Protease Cleavage Non-limiting SEQ ID Site Consensus Sequence Examples NO: Bovine enterokinase DDDDK* DDDDK* 254 Tobacco Etch Virus E P⁵ P⁴YP²Q*(G/S), ENLYFQ*G 255 (TEV) where P², P⁴ and P⁵ can be ENLYFQ*S 256 any amino acid ENIYTQ*G 257 ENIYTQ*S 258 ENIYLQ*G 259 ENIYLQ*S 260 ENVYFQ*G 261 ENVYSQ*S 262 ENVYSQ*G 263 ENVYSQ*S 264 Human Rhinovirus 3C P⁵P⁴LFQ*GP EALFQ*GP 265 where P⁴ is G, A, V, L, I, EVLFQ*GP 266 M, S or T and P⁵ can any ELLFQ*GP 267 amino acid, with D or E DALFQ*GP 268 preferred. DVLFQ*GP 269 DLLFQ*GP 270 SUMO/ULP-1 Tertiary structure polypeptide-G* 271 Thrombin P³P²(R/K)*P^(1′), GVR*G 272 where P³ is any amino acid SAR*G 273 and P² or P^(1′ )is G with the SLR*G 274 other position being any DGR*I 275 amino acid QGK*I 276 Thrombin P⁴P³(R/K)*P^(1′)P^(2′) LVPR*GS 277 where P^(1′ )and P^(2′ )can be any LVPK*GS 278 amino acid except for acidic FIPR*TF 279 amino acids like D or E; and VLPR*SF 280 P³ and P⁴ are hydrophobic IVPR*SF 281 amino acids like F, L, I, Y, IVPR*GY 282 W, V, M, P, C or A VVPR*GV 283 VLPR*LI 284 VMPR*SL 285 MFPR*SL 286 Coagulation Factor I(E/D)GR* IDGR* 287 Xa IEGR* 288 An asterisks (*) indicates the peptide bond that is cleaved by the indicated protease.

It is envisioned that an exogenous protease cleavage site of any and all lengths can be useful in aspects of the present invention with the proviso that the exogenous protease cleavage site is capable of being cleaved by its respective protease. Thus, in aspects of this embodiment, an exogenous protease cleavage site can be, e.g., at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, at least 25 amino acids in length, at least 30 amino acids in length, at least 40 amino acids in length, at least 50 amino acids in length or at least 60 amino acids in length. In other aspects of this embodiment, an exogenous protease cleavage site can be, e.g., at most 6 amino acids in length, at most 7 amino acids in length, at most 8 amino acids in length, at most 9 amino acids in length, at most 10 amino acids in length, at most 15 amino acids in length, at most 20 amino acids in length, at most 25 amino acids in length, at most 30 amino acids in length, at most 40 amino acids in length, at most 50 amino acids in length or at most 60 amino acids in length.

In aspects of this embodiment, a di-chain loop region can be modified to substitute a naturally-occurring protease cleavage site for an exogenous protease cleavage site. In this type of modification, the naturally-occurring protease cleavage site is made inoperable and thus can not be cleaved by its protease. Only the exogenous protease cleavage site can be cleaved by its corresponding exogenous protease. In this type of modification, the exogenous protease site is operably-linked in-frame to a multivalent Clostridial toxin as a fusion protein and the site can be cleaved by its respective exogenous protease. As a non-limiting example, a single-chain modified BoNT/A comprising an exogenous protease cleavage site in the di-chain loop region can be cleaved by its respective exogenous protease to produce the di-chain form of the toxin.

In other aspects of this embodiment, a di-chain loop region can be modified to include an exogenous protease cleavage site in addition to the naturally-occurring protease cleavage site. In this type of modification, both cleavage sites are operably-linked in-frame to a multivalent Clostridial toxin as a fusion protein and both sites can be cleaved by their respective proteases. As a non-limiting example, a single-chain modified BoNT/A that comprises a di-chain loop containing both the naturally-occurring BoNT/A di-chain loop protease cleavage site and an exogenous protease cleavage site can be cleaved by either the naturally occurring di-chain loop protease or by the appropriate exogenous protease to produce the di-chain form of the toxin.

A naturally-occurring protease cleavage site can be made inoperable by altering at least the two amino acids flanking the peptide bond cleaved by the naturally-occurring di-chain loop protease. More extensive alterations can be made, with the proviso that the two cysteine residues of the di-chain loop region remain intact and can still form the disulfide bridge. Non-limiting examples of an amino acid alteration include deletion of an amino acid or replacement of the original amino acid with a different amino acid. Thus, in one embodiment, a naturally-occurring protease cleavage site is made inoperable by altering the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease. In other aspects of this embodiment, a naturally-occurring protease cleavage site is made inoperable by altering, e.g., at least three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at least 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.

In still other aspects of this embodiment, a naturally-occurring di-chain protease cleavage site is made inoperable by altering, e.g., at most three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at most 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.

In an embodiment, an exogenous protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In aspects of this embodiment, a multivalent Clostridial toxin comprises an exogenous protease cleavage site comprises, e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C protease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site or a Factor Xa protease cleavage site. In other aspects of this embodiment, an exogenous protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In an aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a bovine enterokinase cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a bovine enterokinase protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 254. Is still other aspects of this embodiment, a bovine enterokinase protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a Tobacco Etch Virus protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263 or SEQ ID NO: 264. Is still other aspects of this embodiment, a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In still another aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a Human Rhinovirus 3C protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a Human Rhinovirus 3C protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269 or SEQ ID NO: 270. Is still other aspects of this embodiment, a Human Rhinovirus 3C protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In yet another aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a SUMO/ULP-1 protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a SUMO/ULP-1 protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 271. Is still other aspects of this embodiment, a SUMO/ULP-1 protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In a further aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a Thrombin protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a Thrombin protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285 or SEQ ID NO: 286. Is still other aspects of this embodiment, a Thrombin protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavage site can be, e.g., a Coagulation Factor Xa protease cleavage site is located within the di-chain loop of a multivalent Clostridial toxin. In other aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a Coagulation Factor Xa protease cleavage site located within the di-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO: 287 or SEQ ID NO: 288. Is still other aspects of this embodiment, a Coagulation Factor Xa protease cleavage site is located within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another embodiment, an exogenous protease site comprises a Clostridial toxin substrate cleavage site. As used herein, the term “Clostridial toxin substrate cleavage site” means a scissile bond together with adjacent or non-adjacent recognition elements, or both, sufficient for detectable proteolysis at the scissile bond by a Clostridial toxin under conditions suitable for Clostridial toxin protease activity. By definition, a Clostridial toxin substrate cleavage site is susceptible to cleavage by at least one Clostridial toxin under conditions suitable for Clostridial toxin protease activity. Non-limiting examples of Clostridial toxin substrate cleavage site are disclosed in, e.g., Steward, L. E. et al., Self-Activating Clostridial Toxins, U.S. Patent Application 60/718,616 (Sep. 19, 2005).

It is understood that a multivalent Clostridial toxin disclosed in the present specification can optionally include one or more additional components. As a non-limiting example of an optional component, a multivalent Clostridial toxin can further comprise a flexible region comprising a flexible spacer. Non-limiting examples of a flexible spacer include, e.g., a G-spacer GGGGS (SEQ ID NO: 289) or an A-spacer EAAAK (SEQ ID NO: 290). A flexible region comprising flexible spacers can be used to adjust the length of a polypeptide region in order to optimize a characteristic, attribute or property of a polypeptide. Such a flexible region is operably-linked in-frame to the multivalent Clostridial toxin as a fusion protein. As a non-limiting example, a polypeptide region comprising one or more flexible spacers in tandem can be use to better expose a protease cleavage site thereby facilitating cleavage of that site by a protease. As another non-limiting example, a polypeptide region comprising one or more flexible spacers in tandem can be use to better present an enhanced targeting domain, thereby facilitating the binding of that enhanced targeting domain to its receptor.

Thus, in an embodiment, a multivalent Clostridial toxin disclosed in the present specification can further comprise a flexible region comprising a flexible spacer. In another embodiment, a multivalent Clostridial toxin disclosed in the present specification can further comprise flexible region comprising a plurality of flexible spacers in tandem. In aspects of this embodiment, a flexible region can comprise in tandem, e.g., at least 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4 G-spacers or at least 5 G-spacers. In other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at most 1 G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5 G-spacers. In still other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers, at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. In still other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3 A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise a flexible region comprising one or more copies of the same flexible spacers, one or more copies of different flexible-spacer regions, or any combination thereof.

In aspects of this embodiment, a multivalent Clostridial toxin comprising a flexible spacer can be, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

It is envisioned that a multivalent Clostridial toxin disclosed in the present specification can comprise a flexible spacer in any and all locations with the proviso that multivalent Clostridial toxin is capable of performing the intoxication process. In aspects of this embodiment, a flexible spacer is positioned between, e.g., an enzymatic domain and a translocation domain, an enzymatic domain and a binding domain, an enzymatic domain and a protease cleavage site. In other aspects of this embodiment, a G-spacer is positioned between, e.g., an enzymatic domain and a translocation domain, an enzymatic domain and a binding domain, an enzymatic domain and a protease cleavage site. In other aspects of this embodiment, a A-spacer is positioned between, e.g., an enzymatic domain and a translocation domain, an enzymatic domain and a binding domain, an enzymatic domain and a protease cleavage site.

In other aspects of this embodiment, a flexible spacer is positioned between, e.g., a binding domain and a translocation domain, a binding domain and an enzymatic domain, a binding domain and a protease cleavage site. In other aspects of this embodiment, a G-spacer is positioned between, e.g., a binding domain and a translocation domain, a binding domain and an enzymatic domain, a binding domain and a protease cleavage site. In other aspects of this embodiment, a A-spacer is positioned between, e.g., a binding domain and a translocation domain, a binding domain and an enzymatic domain, a binding domain and a protease cleavage site.

In yet other aspects of this embodiment, a flexible spacer is positioned between, e.g., a translocation domain and an enzymatic domain, an translocation domain and a binding domain, an translocation domain and a protease cleavage site. In other aspects of this embodiment, a G-spacer is positioned between, e.g., a translocation domain and an enzymatic domain, an translocation domain and a binding domain, an translocation domain and a protease cleavage site. In other aspects of this embodiment, a A-spacer is positioned between, e.g., a translocation domain and an enzymatic domain, an translocation domain and a binding domain, a translocation domain and a protease cleavage site.

It is envisioned that a multivalent Clostridial toxin disclosed in the present specification can comprise a binding domain 1 and a binding domain 2 in any and all locations with the proviso that multivalent Clostridial toxin is capable of performing the intoxication process. Non-limiting examples are depicted in FIG. 4), FIG. 5, and FIG. 6. The enzymatic domain of naturally-occurring Clostridial toxins contains the native start methionine. Thus, in domain organizations where the enzymatic domain is not in the amino-terminal location an amino acid sequence comprising the start methionine should be placed in front of the amino-terminal domain. Likewise, where a binding domain is in the amino-terminal position, an amino acid sequence comprising a start methionine and a protease cleavage site may be operably-linked in situations in which the enhanced targeting domain requires a free amino terminus, see, e.g., Shengwen Li et al., Degradable Clostridial Toxins, International Patent Application Publication WO 2006/026780 (Mar. 9, 2006). In addition, it is known in the art that when adding a polypeptide that is operably-linked to the amino terminus of another polypeptide comprising the start methionine that the original methionine residue can be deleted.

Thus, in an embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, a translocation domain, a binding domain 2 and an enzymatic domain. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, a translocation domain, a binding domain 2, a protease cleavage site and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, a translocation domain, a binding domain 2, an endogenous protease cleavage site and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, a translocation domain, a binding domain 2, an exogenous protease cleavage site and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a binding domain 1, a translocation domain and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a protease cleavage site, a binding domain 1, a translocation domain and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an endogenous protease cleavage site, a binding domain 1, a translocation domain and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an exogenous protease cleavage site, a binding domain 1, a translocation domain and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 1 and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a protease cleavage site, a translocation domain, a binding domain 1 and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an endogenous protease cleavage site, a translocation domain, a binding domain 1 and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an exogenous protease cleavage site, a translocation domain, a binding domain 1 and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 2 and a binding domain 1. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a protease cleavage site, a translocation domain, a binding domain 2 and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an endogenous protease cleavage site, a translocation domain, a binding domain 2 and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, an exogenous protease cleavage site, a translocation domain, a binding domain 2 and a binding domain 1.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, an enzymatic domain, a translocation domain and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, an enzymatic domain, a protease cleavage site, a translocation domain and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, an enzymatic domain, an endogenous protease cleavage site, a translocation domain and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 1, an enzymatic domain, an exogenous protease cleavage site, a translocation domain and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, a binding domain 1 and an enzymatic domain. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, a protease cleavage site, a binding domain 1 and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an endogenous protease cleavage site, a binding domain 1 and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an exogenous protease cleavage site, a binding domain 1 and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an enzymatic domain and a binding domain 1. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, a protease cleavage site, an enzymatic domain and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an endogenous protease cleavage site, an enzymatic domain and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a translocation domain, a binding domain 2, an exogenous protease cleavage site, an enzymatic domain and a binding domain 1.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 2, a translocation domain, a binding domain 1 and an enzymatic domain. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 2, a translocation domain, a protease cleavage site, a binding domain 1 and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 2, a translocation domain, an endogenous cleavage protease site, a binding domain 1 and an enzymatic domain. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising a binding domain 2, a translocation domain, an exogenous protease cleavage site, a binding domain 1 and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 1 and a binding domain 2. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first protease cleavage site, a translocation domain, a binding domain 1, a second protease cleavage site, and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first endogenous protease cleavage site, a translocation domain, a binding domain 1, a second endogenous protease cleavage site, and a binding domain 2. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first exogenous protease cleavage site, a translocation domain, a binding domain 1, a second exogenous protease cleavage site, and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, a binding domain 2 and a binding domain 1. In an aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first protease cleavage site, a translocation domain, a binding domain 2, a second protease cleavage site, and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first endogenous protease cleavage site, a translocation domain, a binding domain 2, a second endogenous protease cleavage site, and a binding domain 1. In another aspect of this embodiment, a multivalent Clostridial toxin can comprise an amino to carboxyl linear organization comprising an enzymatic domain, a first exogenous protease cleavage site, a translocation domain, a binding domain 2, a second exogenous protease cleavage site, and a binding domain 1.

Aspects of the present invention provide, in part multivalent Clostridial toxins. Non-limiting examples of Clostridial toxin modifications disclosed in the present specification include, e.g., addition of a binding domain 1, addition of a binding domain 2, addition of a protease cleavage site, rearrangement of the enzymatic, translocation and binding domains and addition of a spacer region. It is understood that all such modifications do not substantially affect the ability of a multivalent Clostridial toxin to intoxicate a cell. As used herein, the term “do not substantially affect” means a multivalent Clostridial toxin can still execute the overall cellular mechanism whereby a Clostridial toxin enters a neuron and inhibits neurotransmitter release and encompasses the binding of a Clostridial toxin to a low or high affinity receptor complex, the internalization of the toxin/receptor complex, the translocation of the Clostridial toxin light chain into the cytoplasm and the enzymatic modification of a Clostridial toxin substrate. In aspects of this embodiment, the multivalent Clostridial toxin is, e.g., at least 10% as toxic as a naturally-occurring Clostridial toxin, at least 20% as toxic as a naturally-occurring Clostridial toxin, at least 30% as toxic as a naturally-occurring Clostridial toxin, at least 40% as toxic as a naturally-occurring Clostridial toxin, at least 50% as toxic as a naturally-occurring Clostridial toxin, at least 60% as toxic as a naturally-occurring Clostridial toxin, at least 70% as toxic as a naturally-occurring Clostridial toxin, at least 80% as toxic as a naturally-occurring Clostridial toxin, at least 90% as toxic as a naturally-occurring Clostridial toxin or at least 95% as toxic as a naturally-occurring Clostridial toxin. In aspects of this embodiment, the multivalent Clostridial toxin is, e.g., at most 10% as toxic as a naturally-occurring Clostridial toxin, at most 20% as toxic as a naturally-occurring Clostridial toxin, at most 30% as toxic as a naturally-occurring Clostridial toxin, at most 40% as toxic as a naturally-occurring Clostridial toxin, at most 50% as toxic as a naturally-occurring Clostridial toxin, at most 60% as toxic as a naturally-occurring Clostridial toxin, at most 70% as toxic as a naturally-occurring Clostridial toxin, at most 80% as toxic as a naturally-occurring Clostridial toxin, at most 90% as toxic as a naturally-occurring Clostridial toxin or at most 95% as toxic as a naturally-occurring Clostridial toxin.

Another aspect of the present invention provides polynucleotide molecules encoding multivalent Clostridial toxins disclosed in the present specification. It is envisioned that any and all multivalent Clostridial toxin disclosed in the present specification can be encoded by a polynucleotide molecule.

Aspects of the present invention provide, in part polynucleotide molecules. As used herein, the term “polynucleotide molecule” is synonymous with “nucleic acid molecule” and means a polymeric form of nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, of any length. It is envisioned that any and all polynucleotide molecules that can encode a multivalent Clostridial toxin disclosed in the present specification can be useful, including, without limitation naturally-occurring and non-naturally-occurring DNA molecules and naturally-occurring and non-naturally-occurring RNA molecules. Non-limiting examples of naturally-occurring and non-naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagmid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Well-established molecular biology techniques that may be necessary to make a polynucleotide molecule encoding a multivalent Clostridial toxin disclosed in the present specification including, but not limited to, procedures involving polymerase chain reaction (PCR) amplification, restriction enzyme reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial transformation, nucleic acid purification, nucleic acid sequencing and recombination-based techniques are routine procedures well within the scope of one skilled in the art and from the teaching herein. Non-limiting examples of specific protocols necessary to make a polynucleotide molecule encoding a multivalent Clostridial toxin are described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a variety of commercially available products useful for making a polynucleotide molecule encoding a multivalent Clostridial toxin are widely available. These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.

Another aspect of the present invention provides a method of producing a multivalent Clostridial toxin disclosed in the present specification, such method comprising the step of expressing a polynucleotide molecule encoding a multivalent Clostridial toxin in a cell. Another aspect of the present invention provides a method of producing a multivalent Clostridial toxin disclosed in the present specification, such method comprising the steps of introducing an expression construct comprising a polynucleotide molecule encoding a multivalent Clostridial toxin into a cell and expressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, a multivalent Clostridial toxin. It is envisioned that any and all multivalent Clostridial toxins disclosed in the present specification can be produced using the methods disclosed in the present specification. It is also envisioned that any and all polynucleotide molecules encoding a multivalent Clostridial toxins disclosed in the present specification can be useful in producing a multivalent Clostridial toxins disclosed in the present specification using the methods disclosed in the present specification.

The methods disclosed in the present specification include, in part, an expression construct. An expression construct comprises a polynucleotide molecule disclosed in the present specification operably-linked to an expression vector useful for expressing the polynucleotide molecule in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a polynucleotide molecule encoding a multivalent Clostridial toxin, including, without limitation, a viral expression vector; a prokaryotic expression vector; eukaryotic expression vectors, such as, e.g., a yeast expression vector, an insect expression vector and a mammalian expression vector; and a cell-free extract expression vector. It is further understood that expression vectors useful to practice aspects of these methods may include those which express a multivalent Clostridial toxin under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viral expression vector operably-linked to a polynucleotide molecule encoding a multivalent Clostridial toxin; a prokaryotic expression vector operably-linked to a polynucleotide molecule encoding a multivalent Clostridial toxin; a yeast expression vector operably-linked to a polynucleotide molecule encoding a multivalent Clostridial toxin; an insect expression vector operably-linked to a polynucleotide molecule encoding a multivalent Clostridial toxin; and a mammalian expression vector operably-linked to a polynucleotide molecule encoding a multivalent Clostridial toxin. Other aspects of this embodiment include, without limitation, expression constructs suitable for expressing a multivalent Clostridial toxin disclosed in the present specification using a cell-free extract comprising a cell-free extract expression vector operably linked to a polynucleotide molecule encoding a multivalent Clostridial toxin.

The methods disclosed in the present specification include, in part, a cell. It is envisioned that any and all cells can be used. Thus, aspects of this embodiment include, without limitation, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium; and eukaryotic cells including, without limitation, yeast strains, such as, e.g., those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and mammalian cells and cell lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human. Cell lines may be obtained from the American Type Culture Collection, European Collection of Cell Cultures and the German Collection of Microorganisms and Cell Cultures. Non-limiting examples of specific protocols for selecting, making and using an appropriate cell line are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press, 3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part, introducing into a cell a polynucleotide molecule. A polynucleotide molecule introduced into a cell can be transiently or stably maintained by that cell. Stably-maintained polynucleotide molecules may be extra-chromosomal and replicate autonomously, or they may be integrated into the chromosomal material of the cell and replicate non-autonomously. It is envisioned that any and all methods for introducing a polynucleotide molecule disclosed in the present specification into a cell can be used. Methods useful for introducing a nucleic acid molecule into a cell include, without limitation, chemical-mediated transfection such as, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated transfection, such as, e.g., biolistic particle delivery, microinjection, protoplast fusion and electroporation; and viral-mediated transfection, such as, e.g., retroviral-mediated transfection, see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). One skilled in the art understands that selection of a specific method to introduce an expression construct into a cell will depend, in part, on whether the cell will transiently contain an expression construct or whether the cell will stably contain an expression construct. These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termed transfection, is used to introduce a polynucleotide molecule encoding a multivalent Clostridial toxin into a cell. In chemical-mediated methods of transfection the chemical reagent forms a complex with the nucleic acid that facilitates its uptake into the cells. Such chemical reagents include, without limitation, calcium phosphate-mediated, see, e.g., Martin Jordan & Florian Worm, Transfection of adherent and suspended cells by calcium phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationic polymer-mediated like polyethyleneimine (PEI)-mediated and polylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine strategies for plasmid delivery to brain-derived cells, 33(2) Methods 144-150 (2004). Such chemical-mediated delivery systems can be prepared by standard methods and are commercially available, see, e.g., CellPhect Transfection Kit (Amersham Biosciences, Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™ Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500 Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect and Effectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is used to introduce a polynucleotide molecule encoding a multivalent Clostridial toxin into a cell. Physical techniques include, without limitation, electroporation, biolistic and microinjection. Biolistics and microinjection techniques perforate the cell wall in order to introduce the nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga et al., Plasmid-mediated gene transfer in neurons using the biolistics technique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien & Sarah C. R. Lummis, Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells, 33(2) Methods 121-125 (2004). Electroporation, also termed electropermeabilization, uses brief, high-voltage, electrical pulses to create transient pores in the membrane through which the nucleic acid molecules enter and can be used effectively for stable and transient transfections of all cell types, see, e.g., M. Golzio et al., In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., New non-viral method for gene transfer into primary cells, 33(2) Methods 151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termed transduction, is used to introduce a polynucleotide molecule encoding a multivalent Clostridial toxin into a cell. In viral-mediated methods of transient transduction, the process by which viral particles infect and replicate in a host cell has been manipulated in order to use this mechanism to introduce a nucleic acid molecule into the cell. Viral-mediated methods have been developed from a wide variety of viruses including, without limitation, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); and Maurizio Federico, From lentiviruses to lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M. Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); Karim Benihoud et al, Adenovirus vectors for gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler, Adeno-associated viral vectors for gene transfer and gene therapy, 380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus and adeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002); Edward A. Burton et al., Gene delivery using herpes simplex virus vectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al., Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); Ilya Frolov et al., Alphavirus-based expression vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996); Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1) Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4) Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann, Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, are often selected for mammalian cell transduction because adenoviruses handle relatively large polynucleotide molecules of about 36 kb, are produced at high titer, and can efficiently infect a wide variety of both dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermens et al., Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci. Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches for generating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction using adenoviral-based system do not support prolonged protein expression because the nucleic acid molecule is carried from an episome in the cell nucleus, rather than being integrated into the host cell chromosome. Adenoviral vector systems and specific protocols for how to use such vectors are disclosed in, e.g., ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™ Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) and AdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene, Inc.

Nucleic acid molecule delivery can also use single-stranded RNA retroviruses, such as, e.g., oncoretroviruses and lentiviruses. Retroviral-mediated transduction often produce transduction efficiencies close to 100%, can easily control the proviral copy number by varying the multiplicity of infection (MOI), and can be used to either transiently or stably transduce cells, see, e.g., Tiziana Tonini et al., Transient production of retroviral- and lentiviral-based vectors for the transduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004); Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); Félix Recillas-Targa, Gene transfer and expression in mammalian cell lines and transgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and Roland Wolkowicz et al., Lentiviral vectors for the delivery of DNA into mammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviral particles consist of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. The retrovirus infects a host cell by injecting its RNA into the cytoplasm along with the reverse transcriptase enzyme. The RNA template is then reverse transcribed into a linear, double stranded cDNA that replicates itself by integrating into the host cell genome. Viral particles are spread both vertically (from parent cell to daughter cells via the provirus) as well as horizontally (from cell to cell via virions). This replication strategy enables long-term persistent expression since the nucleic acid molecules of interest are stably integrated into a chromosome of the host cell, thereby enabling long-term expression of the protein. For instance, animal studies have shown that lentiviral vectors injected into a variety of tissues produced sustained protein expression for more than 1 year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector, 272(5259) Science 263-267 (1996). The Oncoretroviruses-derived vector systems, such as, e.g., Moloney murine leukemia virus (MoMLV), are widely used and infect many different non-dividing cells. Lentiviruses can also infect many different cell types, including dividing and non-dividing cells and possess complex envelope proteins, which allows for highly specific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectors are disclosed in, e.g., U.S. patent Nos. Manfred Gossen & Hermann Bujard, Tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7, 1995) and Hermann Bujard & Manfred Gossen, Methods for regulating gene expression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness, Polynucleotides encoding insect steroid hormone receptor polypeptides and cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996) and David S. Hogness, Polynucleotide encoding insect ecdysone receptor, U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al., Progesterone receptor having C. terminal hormone binding domain truncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23, 1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Pat. No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systems can be prepared by standard methods and are commercially available, see, e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-On Gene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.) and GeneSwitch™ System A Mifepristone-Regulated Expression System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and Complete Control® Retroviral Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.) and Complete Control® Retroviral Inducible Mammalian Expression System Instruction Manual, 064005e.

The methods disclosed in the present specification include, in part, expressing a multivalent Clostridial toxin from a polynucleotide molecule. It is envisioned that any of a variety of expression systems may be useful for expressing a multivalent Clostridial toxin from a polynucleotide molecule disclosed in the present specification, including, without limitation, cell-based systems and cell-free expression systems. Cell-based systems include, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Cell-free systems include, without limitation, wheat germ extracts, rabbit reticulocyte extracts and E. coli extracts and generally are equivalent to the method disclosed herein. Expression of a polynucleotide molecule using an expression system can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, viral-mediated expression, stably-integrated expression, and transient expression. Expression systems that include well-characterized vectors, reagents, conditions and cells are well-established and are readily available from commercial vendors that include, without limitation, Ambion, Inc. Austin Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limiting examples on the selection and use of appropriate heterologous expression systems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh, Expression Systems for Production of Heterologous Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.

A variety of cell-based expression procedures are useful for expressing a multivalent Clostridial toxin encoded by polynucleotide molecule disclosed in the present specification. Examples included, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Viral expression systems include, without limitation, the ViraPower™ Lentiviral (Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems (Invitrogen, Inc., Carlsbad, Calif.), the AdEasy™ XL Adenoviral Vector System (Stratagene, La Jolla, Calif.) and the ViraPort® Retroviral Gene Expression System (Stratagene, La Jolla, Calif.). Non-limiting examples of prokaryotic expression systems include the Champion™ pET Expression System (EMD Biosciences-Novagen, Madison, Wis.), the TriEx™ Bacterial Expression System (EMD Biosciences-Novagen, Madison, Wis.), the QIAexpress® Expression System (QIAGEN, Inc.), and the Affinity® Protein Expression and Purification System (Stratagene, La Jolla, Calif.). Yeast expression systems include, without limitation, the EasySelect™ Pichia Expression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo™ Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and the SpECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad, Calif.). Non-limiting examples of baculoviral expression systems include the BaculoDirect™ (Invitrogen, Inc., Carlsbad, Calif.), the Bac-to-Bac® (Invitrogen, Inc., Carlsbad, Calif.), and the BD BaculoGold™ (BD Biosciences-Pharmigen, San Diego, Calif.). Insect expression systems include, without limitation, the Drosophila Expression System (DES®) (Invitrogen, Inc., Carlsbad, Calif.), InsectSelect™ System (Invitrogen, Inc., Carlsbad, Calif.) and InsectDirect™ System (EMD Biosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalian expression systems include the T-REx™ (Tetracycline-Regulated Expression) System (Invitrogen, Inc., Carlsbad, Calif.), the Flp-In™ T-REx™ System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA™ system (Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen, Inc., Carlsbad, Calif.), the Exchanger® System, InterPlay™ Mammalian TAP System (Stratagene, La Jolla, Calif.), Complete Control® Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.) and LacSwitch® II Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.).

Another procedure of expressing a multivalent Clostridial toxin encoded by polynucleotide molecule disclosed in the present specification employs a cell-free expression system such as, without limitation, prokaryotic extracts and eukaryotic extracts. Non-limiting examples of prokaryotic cell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science, Indianapolis, Ind.), the ActivePro In Vitro Translation Kit (Ambion, Inc., Austin, Tex.), the EcoPro™ System (EMD Biosciences-Novagen, Madison, Wis.) and the Expressway™ Plus Expression System (Invitrogen, Inc., Carlsbad, Calif.). Eukaryotic cell extract include, without limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science, Indianapolis, Ind.), the TnT® Coupled Wheat Germ Extract Systems (Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit (Ambion, Inc., Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc., Austin, Tex.), the PROTEINscript® II System (Ambion, Inc., Austin, Tex.) and the TnT® Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wis.).

Aspects of the present invention can also be described as follows:

-   1. A polypeptide comprising an endopeptidase domain; a translocation     domain effective to facilitate the movement of said endopeptidase     domain across an endosomal membrane; and at least two binding     domains, wherein: a first binding domain comprises a first ligand     binding, under physiological conditions, to a first cell surface     receptor displayed by the target cell; and a second binding domain     comprises a second ligand binding, under physiological conditions,     to a second cell surface receptor displayed by the target cell;     wherein said target cell internalizes said polypeptide upon binding     of said polypeptide to said target cell. -   2. The polypeptide of 1 wherein said second cell surface receptor     comprises a caveolin-binding domain. -   3. The polypeptide of 1 wherein said second cell surface receptor     comprises TNFR-1. -   4. The polypeptide of 3 wherein said second ligand comprises a     TNFR-1 binding protein selected from the group consisting of TNF-α     and a TNF-α derivative comprising a TNFR-1-binding domain. -   5. The polypeptide of 2 wherein said second ligand binds a glycosyl     phosphatylinositol (GPI)-linked membrane protein. -   6. The polypeptide of 1 wherein said second cell surface receptor     comprises an antibody variable (V) domain region. -   7. The polypeptide of 6 wherein said second ligand comprises an     antigen that selectively binds the antibody variable domain region     of said second cell surface receptor. -   8. The polypeptide of 1 wherein said second cell surface receptor     directly binds clatherin. -   9. The polypeptide of 1 wherein said second cell surface receptor     indirectly binds clatherin. -   10. The polypeptide of 9 wherein the second cell surface receptor     has the amino acid sequence tyrosine-X-arginine-phenylalanine near     the carboxy terminus. -   11. The polypeptide of 9 wherein the second cell surface receptor     binds an adaptin. -   12. The polypeptide of claim 11 wherein the second cell surface     receptor binds Adaptor Protein-2. -   13. The polypeptide of 1 wherein the first ligand selectively binds     a Clostridial neurotoxin-binding cell surface receptor. -   14. The polypeptide of 13 wherein the first ligand comprises a     Clostridial neurotoxin binding domain. -   15. The polypeptide of 15 wherein the first ligand comprises a     Clostridial neurotoxin binding domain selected from the group     consisting of a TeNT receptor binding domain, a BoNT-A receptor     binding domain, a BoNT-B receptor binding domain, a BoNT-C receptor     binding domain, a BoNT-C1 receptor binding domain, a BoNT-D receptor     binding domain, a BoNT-E receptor binding domain, a BoNT-F receptor     binding domain, a BoNT-G receptor binding domain HA or NTNH     β-trefoil domain of a neurotoxin associated protein (NAP) associated     with any Clostridial neurotoxin, and variants and isoforms of any of     the above. -   16. The polypeptide of 1 wherein first or second ligand comprises a     receptor-binding domain selected from the group consisting of: a     nerve growth factor (NGF) receptor binding domain; a leukemia     inhibitory factor (LIF) receptor binding domain; a basic fibroblast     growth factor (bFGF) receptor binding domain; a brain-derived     neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3     (NT-3) receptor binding domain; a hydra head activator peptide     (HHAP) receptor binding domain; a transforming growth factor 1     (TGF-1) receptor binding domain; a transforming growth factor 2     (TGF-2) receptor binding domain; a transforming growth factor 3     (TGF-3) receptor binding domain; an epidermal growth factor (EGF)     receptor binding domain; a ciliary neurotrophic factor (CNTF)     receptor binding domain; a tumor necrosis factor (TNF-) receptor     binding domain; an interleukin-1 (IL-1) receptor binding domain; an     interleukin-1 (IL-1) receptor binding domain; an interleukin-8     (IL-8) receptor binding domain; a bradykinin receptor binding     domain; a dynorphin receptor binding domain; a β-endorphin receptor     binding domain; an etorphine receptor binding domain; an     endomorphin-1 receptor binding domain; an endomorphin-2 receptor     binding domain; a leu-enkephalin receptor binding domain; a     met-enkephalin receptor binding domain; a galanin receptor binding     domain; a lofentanil receptor binding domain; a nociceptin receptor     binding domain; an antigen-binding variable region of an antibody     against the lactoseries carbohydrate epitopes found on the surface     of dorsal root ganglion neurons; an antigen-binding variable region     of an antibody binding any of the receptors for the binding domains     given above and an antibody against the surface expressed antigen     Thyl. -   17. The polypeptide of 16 wherein said first and second cell surface     receptors are identical. -   18. The polypeptide of 17 wherein the first and second ligands are     identical. -   19. The polypeptide of 16 wherein said first and second ligands are     different. -   20. The polypeptide of 15 wherein said second ligand comprises a     receptor-binding domain selected from the group consisting of: a     nerve growth factor (NGF) receptor binding domain; a leukemia     inhibitory factor (LIF) receptor binding domain; a basic fibroblast     growth factor (bFGF) receptor binding domain; a brain-derived     neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3     (NT-3) receptor binding domain; a hydra head activator peptide     (HHAP) receptor binding domain; a transforming growth factor 1     (TGF-1) receptor binding domain; a transforming growth factor 2     (TGF-2) receptor binding domain; a transforming growth factor 3     (TGF-3) receptor binding domain; an epidermal growth factor (EGF)     receptor binding domain; a ciliary neurotrophic factor (CNTF)     receptor binding domain; a tumor necrosis factor (TNF-) receptor     binding domain; an interleukin-1 (IL-1) receptor binding domain; an     interleukin-1 (IL-1) receptor binding domain; an interleukin-8     (IL-8) receptor binding domain; a bradykinin receptor binding     domain; a dynorphin receptor binding domain; a β-endorphin receptor     binding domain; an etorphine receptor binding domain; an     endomorphin-1 receptor binding domain; an endomorphin-2 receptor     binding domain; a leu-enkephalin receptor binding domain; a     met-enkephalin receptor binding domain; a galanin receptor binding     domain; a lofentanil receptor binding domain; a nociceptin receptor     binding domain; an antigen-binding variable region of an antibody     against the lactoseries carbohydrate epitopes found on the surface     of dorsal root ganglion neurons; an antigen-binding variable region     of an antibody binding any of the receptors for the binding domains     given above and an antibody against the surface expressed antigen     Thyl. -   21. The polypeptide of 16 wherein said second cell surface receptor     comprises a caveolin-binding domain. -   22. The polypeptide of 16 wherein said second cell surface receptor     comprises TNFR-1. -   23. The polypeptide of 22 wherein said second ligand comprises a     TNFR-1 binding protein selected from the group consisting of TNF-α     and a TNF-α derivative comprising a TNFR-1-binding domain. -   24. The polypeptide of 21 wherein said second ligand binds a     glycosyl phosphatylinositol (GPI)-linked membrane protein. -   25. The polypeptide of 16 wherein said second cell surface receptor     comprises an antibody variable (V) domain region. -   26. The polypeptide of 25 wherein said second ligand comprises an     antigen that selectively binds the antibody variable domain region     of said second cell surface receptor. -   27. The polypeptide of 16 wherein said second cell surface receptor     directly or indirectly binds clatherin. -   28. The polypeptide of 27 wherein said second cell surface receptor     indirectly binds clatherin. -   29. The polypeptide of 27 wherein the second cell surface receptor     has the amino acid sequence tyrosine-X-arginine-phenylalanine near     the carboxy terminus. -   30. The polypeptide of 27 wherein the second cell surface receptor     directly binds an adaptin. -   31. The polypeptide of 30 wherein the second cell surface receptor     binds Adaptor Protein-2. -   32. A method for inhibiting or decreasing exocytosis or secretion in     a target cell comprising contacting said target cell under     physiological conditions with an effective dose of a multivalent     Clostridial toxin composition comprising: an endopeptidase domain; a     translocation domain effective to facilitate the movement of said     endopeptidase domain from an endocytotic vesicle to the cytoplasm of     said target cell; and at least two binding domains, wherein: a first     binding domain comprises a first ligand selectively binding under     physiological conditions to a first cell surface receptor     selectively displayed by the target cell; and a second binding     domain comprises a second ligand binding, under physiological     conditions, to a second cell surface receptor displayed by the     target cell; wherein said endopeptidase domain catalyzes cleavage of     a intracellular SNARE protein within the target cell, thereby     inhibiting or decreasing exocytosis or secretion by said cell. -   33. The method of 32 wherein said second cell surface receptor     comprises a caveolin-binding domain. -   34. The method of 32 wherein said second cell surface receptor     comprises TNFR-1. -   35. The method of 34 wherein said second ligand comprises a TNFR-1     binding protein selected from the group consisting of TNF-α and a     TNF-α derivative comprising a TNFR-1-binding domain. -   36. The method of 33 wherein said second ligand binds a glycosyl     phosphatylinositol (GPI)-linked membrane protein. -   37. The method of 32 wherein said second cell surface receptor     comprises an antibody variable (V) domain region. -   38. The method of 37 wherein said second ligand comprises an antigen     that selectively binds the antibody variable domain region of said     second cell surface receptor. -   39. The method of 32 wherein said second cell surface receptor     directly binds clatherin. -   40. The method of 32 wherein said second cell surface receptor     indirectly binds clatherin. -   41. The method of 39 wherein the second cell surface receptor has     the amino acid sequence tyrosine-X-arginine-phenylalanine near the     carboxy terminus. -   42. The method of 39 wherein the second cell surface receptor binds     an adaptin. -   43. The method of 42 wherein the second cell surface receptor binds     Adaptor Protein-2. -   44. The method of 32 wherein the first ligand selectively binds a     Clostridial neurotoxin-binding cell surface receptor. -   45. The method of 44 wherein the first ligand comprises a     clostridial neurotoxin binding domain. -   46. The method of 45 wherein the first ligand comprises a     clostridial neurotoxin binding domain selected from the group     consisting of a TeNT receptor binding domain, a BoNT-A receptor     binding domain, a BoNT-B receptor binding domain, a BoNT-C receptor     binding domain, a BoNT-C1 receptor binding domain, a BoNT-D receptor     binding domain, a BoNT-E receptor binding domain, a BoNT-F receptor     binding domain, a BoNT-G receptor binding domain HA or NTNH β     trefoil domain of a neurotoxin associated protein (NAP) associated -   47. The method of 32 wherein first or second ligand comprises a     receptor-binding domain selected from the group consisting of: a     nerve growth factor (NGF) receptor binding domain; a leukemia     inhibitory factor (LIF) receptor binding domain; a basic fibroblast     growth factor (bFGF) receptor binding domain; a brain-derived     neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3     (NT-3) receptor binding domain; a hydra head activator peptide     (HHAP) receptor binding domain; a transforming growth factor 1     (TGF-1) receptor binding domain; a transforming growth factor 2     (TGF-2) receptor binding domain; a transforming growth factor 3     (TGF-3) receptor binding domain; an epidermal growth factor (EGF)     receptor binding domain; a ciliary neurotrophic factor (CNTF)     receptor binding domain; a tumor necrosis factor (TNF-) receptor     binding domain; an interleukin-1 (IL-1) receptor binding domain; an     interleukin-1 (IL-1) receptor binding domain; an interleukin-8     (IL-8) receptor binding domain; a bradykinin receptor binding     domain; a dynorphin receptor binding domain; a β-endorphin receptor     binding domain; an etorphine receptor binding domain; an     endomorphin-1 receptor binding domain; an endomorphin-2 receptor     binding domain; a leu-enkephalin receptor binding domain; a     met-enkephalin receptor binding domain; a galanin receptor binding     domain; a lofentanil receptor binding domain; a nociceptin receptor     binding domain; an antigen-binding variable region of an antibody     against the lactoseries carbohydrate epitopes found on the surface     of dorsal root ganglion neurons; an antigen-binding variable region     of an antibody binding any of the receptors for the binding domains     given above and an antibody against the surface expressed antigen     Thyl. -   48. The method of 47 wherein said first and second cell surface     receptors are identical. -   49. The method of 48 wherein the first and second ligands are     identical. -   50. The method of 47 wherein said first and second ligands are     different. -   51. The method of 46 wherein said second ligand comprises a     receptor-binding domain selected from the group consisting of: a     nerve growth factor (NGF) receptor binding domain; a leukemia     inhibitory factor (LIF) receptor binding domain; a basic fibroblast     growth factor (bFGF) receptor binding domain; a brain-derived     neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3     (NT-3) receptor binding domain; a hydra head activator peptide     (HHAP) receptor binding domain; a transforming growth factor 1     (TGF-1) receptor binding domain; a transforming growth factor 2     (TGF-2) receptor binding domain; a transforming growth factor 3     (TGF-3) receptor binding domain; an epidermal growth factor (EGF)     receptor binding domain; a ciliary neurotrophic factor (CNTF)     receptor binding domain; a tumor necrosis factor (TNF-) receptor     binding domain; an interleukin-1 (IL-1) receptor binding domain; an     interleukin-1 (IL-1) receptor binding domain; an interleukin-8     (IL-8) receptor binding domain; a bradykinin receptor binding     domain; a dynorphin receptor binding domain; β-endorphin receptor     binding domain; an etorphine receptor binding domain; an     endomorphin-1 receptor binding domain; an endomorphin-2 receptor     binding domain; a leu-enkephalin receptor binding domain; a     met-enkephalin receptor binding domain; a galanin receptor binding     domain; a lofentanil receptor binding domain; a nociceptin receptor     binding domain; an antigen-binding variable region of an antibody     against the lactoseries carbohydrate epitopes found on the surface     of dorsal root ganglion neurons; an antigen-binding variable region     of an antibody binding any of the receptors for the binding domains     given above and an antibody against the surface expressed antigen     Thyl. -   52. The method of 47 wherein said second cell surface receptor     comprises a caveolin-binding domain. -   53. The method of 46 wherein said second cell surface receptor     comprises TNFR-1. -   54. The method of 53 wherein said second ligand comprises a TNFR-1     binding protein selected from the group consisting of TNF-α and a     TNF-α derivative comprising a TNFR-1-binding domain. -   55. The method of 52 wherein said second ligand binds a glycosyl     phosphatylinositol (GPI)-linked membrane protein. -   56. The method of 16 wherein said second cell surface receptor     comprises an antibody variable (V) domain region. -   57. The method of 56 wherein said second ligand comprises an antigen     that selectively binds the antibody variable domain region of said     second cell surface receptor. -   58. The method of 47 wherein said second cell surface receptor     directly or indirectly binds clatherin. -   59. The method of 58 wherein said second cell surface receptor     indirectly binds clatherin. -   60. The method of 58 wherein the second cell surface receptor has     the amino acid sequence tyrosine-X-arginine-phenylalanine near the     carboxy terminus. -   61. The method of 58 wherein the second cell surface receptor     directly binds an adaptin. -   62. The method of 61 wherein the second cell surface receptor binds     Adaptor Protein-2. -   63. The method of 32 wherein said exocytosis or secretion is     associated with a pathological condition related to a condition     selected from the group consisting of a movement disorder, a     disorder of the sensory nervous system, acute or chronic pain,     cancer, pancreatitis, hyperhydrosis, glandular disorders, viral     infections, and cystic fibrosis. -   64. The method of 32 wherein the multivalent Clostridial toxin is     administered by injection. -   65. A nucleic acid molecule encoding a polypeptide according to any     one of 1-31.

Aspects of the present invention can also be described as follows:

-   1. A multivalent Clostridial toxin comprising:     -   a) a Clostridial toxin enzymatic domain capable of executing an         enzymatic target modification step of a Clostridial toxin         intoxication process;     -   b) a Clostridial toxin translocation domain capable of executing         a translocation step of a Clostridial toxin intoxication         process;     -   c) a first binding domain capable of executing a cell binding         step of a Clostridial toxin intoxication process by selectively         binding a first cell surface receptor displayed by the target         cell; and     -   d) a second binding domain capable of executing a cell binding         step of a Clostridial toxin intoxication process by selectively         binding a second cell surface receptor displayed by the target         cell; and     -   e) a protease cleavage site, wherein cleavage of the protease         cleavage site converts the single-chain form of the modified         Clostridial toxin into the di-chain form. -   2. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises amino to carboxyl linear     organization comprising a binding domain 1, a translocation domain,     a binding domain 2, a protease cleavage site and an enzymatic     domain. -   3. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising an enzymatic domain, a protease cleavage     site, a binding domain 1, a translocation domain and a binding     domain 2 -   4. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising an enzymatic domain, a protease cleavage     site, a translocation domain, a binding domain 1 and a binding     domain 2 -   5. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising an enzymatic domain, a protease cleavage     site, a translocation domain, a binding domain 2 and a binding     domain 1 -   6. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising a binding domain 1, an enzymatic domain, a     protease cleavage site, a translocation domain and a binding domain     2 -   7. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises amino to carboxyl linear     organization comprising a translocation domain, a binding domain 2,     a protease cleavage site, a binding domain 1 and an enzymatic domain -   8. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising a translocation domain, a binding domain 2,     a protease cleavage site, an enzymatic domain and a binding domain 1 -   9. The multivalent Clostridial toxin according to 1, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising a binding domain 2, a translocation domain,     a protease cleavage site, a binding domain 1 and an enzymatic domain -   10. The multivalent Clostridial toxin according to 1, wherein the     protease cleavage site is an endogenous protease cleavage site or an     exogenous protease cleavage site. -   11. The multivalent Clostridial toxin according to 10, wherein the     endogenous protease cleavage site is selected from the group     consisting of a BoNT/A di-chain loop protease cleavage site, a     BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop     protease cleavage site, a BoNT/D di-chain loop protease cleavage     site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F     di-chain loop protease cleavage site, a BoNT/G di-chain loop     protease cleavage site and a TeNT di-chain loop protease cleavage     site. -   12. The multivalent Clostridial toxin according to 10, wherein the     exogenous protease cleavage site is selected from the group     consisting of a bovine enterokinase protease cleavage site, a     Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C     protease cleavage site, a SUMO/ULP-1 protease cleavage site, a     Thrombin protease cleavage site, and a Factor Xa protease cleavage     site. -   13. A multivalent Clostridial toxin comprising:     -   a) a Clostridial toxin enzymatic domain capable of executing an         enzymatic target modification step of a Clostridial toxin         intoxication process;     -   b) a Clostridial toxin translocation domain capable of executing         a translocation step of a Clostridial toxin intoxication         process;     -   c) a first binding domain capable of executing a cell binding         step of a Clostridial toxin intoxication process by selectively         binding a first cell surface receptor displayed by the target         cell;     -   d) a second binding domain capable of executing a cell binding         step of a Clostridial toxin intoxication process by selectively         binding a second cell surface receptor displayed by the target         cell;     -   e) a first protease cleavage site, wherein cleavage of the first         protease cleavage site converts the single-chain form of the         modified Clostridial toxin into the di-chain form; and     -   f) a second protease cleavage site; wherein cleavage of the         second protease causes a structural confirmation in the first         and second binding domains which facilitates the selectively         binding activity of the first binding domain, the second binding         domain, or both the first and second binding domains. -   14. The multivalent Clostridial toxin according to 13, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising an enzymatic domain, a first protease     cleavage site, a translocation domain, a binding domain 1, a second     protease cleavage site and a binding domain 2 -   15. The multivalent Clostridial toxin according to 13, wherein the     multivalent Clostridial toxin comprises an amino to carboxyl linear     organization comprising an enzymatic domain, a first protease     cleavage site, a translocation domain, a binding domain 2, a second     protease cleavage site and a binding domain 1 -   16. The modified Clostridial toxin according to 13, wherein the     first protease cleavage site is an endogenous protease cleavage site     or an exogenous protease cleavage site. -   17. The multivalent Clostridial toxin according to 16, wherein the     endogenous protease cleavage site is selected from the group     consisting of a BoNT/A di-chain loop protease cleavage site, a     BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop     protease cleavage site, a BoNT/D di-chain loop protease cleavage     site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F     di-chain loop protease cleavage site, a BoNT/G di-chain loop     protease cleavage site and a TeNT di-chain loop protease cleavage     site. -   18. The multivalent Clostridial toxin according to 16, wherein the     exogenous protease cleavage site is selected from the group     consisting of a bovine enterokinase protease cleavage site, a     Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C     protease cleavage site, a SUMO/ULP-1 protease cleavage site, a     Thrombin protease cleavage site, and a Factor Xa protease cleavage     site. -   19. The modified Clostridial toxin according to 13, wherein the     second protease cleavage site is an endogenous protease cleavage     site or an exogenous protease cleavage site. -   20. The multivalent Clostridial toxin according to 19, wherein the     endogenous protease cleavage site is selected from the group     consisting of a BoNT/A di-chain loop protease cleavage site, a     BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop     protease cleavage site, a BoNT/D di-chain loop protease cleavage     site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F     di-chain loop protease cleavage site, a BoNT/G di-chain loop     protease cleavage site and a TeNT di-chain loop protease cleavage     site. -   21. The multivalent Clostridial toxin according to 19, wherein the     exogenous protease cleavage site is selected from the group     consisting of a bovine enterokinase protease cleavage site, a     Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C     protease cleavage site, a SUMO/ULP-1 protease cleavage site, a     Thrombin protease cleavage site, and a Factor Xa protease cleavage     site. -   22. The multivalent Clostridial toxin according to either 1 or 13,     wherein the Clostridial toxin enzymatic domain is selected from the     group consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic     domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a     BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G     enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain     and a BuNT enzymatic domain. -   23. The multivalent Clostridial toxin according to 22, wherein the     Clostridial toxin enzymatic domain comprises an amino acid sequence     selected from the group consisting of amino acids 1-448 of SEQ ID     NO: 1, amino acids 1-441 SEQ ID NO: 2, amino acids 1-449 of SEQ ID     NO: 3, amino acids 1-445 of SEQ ID NO: 4, amino acids 1-422 of SEQ     ID NO: 5, amino acids 1-439 of SEQ ID NO: 6, amino acids 1-446 of     SEQ ID NO: 7, amino acid 1-457 of SEQ ID NO: 8, amino acid 1-431 of     SEQ ID NO: 9, and amino acid 1-422 of SEQ ID NO: 10. -   24. The multivalent Clostridial toxin according either 1 or 13,     wherein the Clostridial toxin translocation domain is selected from     the group consisting of a BoNT/A translocation domain, a BoNT/B     translocation domain, a BoNT/C1 translocation domain, a BoNT/D     translocation domain, a BoNT/E translocation domain, a BoNT/F     translocation domain, a BoNT/G translocation domain, a TeNT     translocation domain, a BaNT translocation domain and a BuNT     translocation domain. -   25. The multivalent Clostridial toxin according to 24, wherein the     Clostridial toxin translocation domain comprises an amino acid     sequence selected from the group consisting of amino acids 449-873     of SEQ ID NO: 1, amino acids 442-860 SEQ ID NO: 2, amino acids     450-868 of SEQ ID NO: 3, amino acids 446-864 of SEQ ID NO: 4, amino     acids 423-847 of SEQ ID NO: 5, amino acids 440-866 of SEQ ID NO: 6,     amino acids 447-865 of SEQ ID NO: 7, amino acid 458-881 of SEQ ID     NO: 8, amino acid 432-857 of SEQ ID NO: 9, and amino acid 423-847 of     SEQ ID NO: 10. -   26. The multivalent Clostridial toxin according either 1 or 13,     wherein the first binding domain comprises a binding domain selected     from the group consisting of a Clostridial toxin binding domain, a     Clostridial non-toxin associated protein β-trefoil domain and an FGF     β-trefoil domain. -   27. The multivalent Clostridial toxin according to 26, wherein the     Clostridial toxin binding domain is selected from the group     consisting of a BoNT/A binding domain, a BoNT/B binding domain, a     BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E binding     domain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT     binding domain, a BaNT binding domain, and a BuNT binding domain. -   28. The multivalent Clostridial toxin according to 26, wherein the     Clostridial toxin binding domain comprises a BoNT/A H_(C) binding     domain, a BoNT/B H_(C) binding domain, a BoNT/C1 H_(C) binding     domain, a BoNT/D H_(C) binding domain, a BoNT/E H_(C) binding     domain, a BoNT/F H_(C) binding domain, a BoNT/G H_(C) binding     domain, a TeNT H_(C) binding domain, a BaNT H_(C) binding domain,     and a BuNT H_(C) binding domain. -   29. The multivalent Clostridial toxin according to 28, wherein the     Clostridial toxin binding domain comprises an amino acid sequence     selected from the group consisting of amino acids 874-1296 of SEQ ID     NO: 1, amino acids 861-1291 SEQ ID NO: 2, amino acids 869-1291 of     SEQ ID NO: 3, amino acids 865-1276 of SEQ ID NO: 4, amino acids     848-1252 of SEQ ID NO: 5, amino acids 867-1274 of SEQ ID NO: 6,     amino acids 866-1297 of SEQ ID NO: 7, amino acid 882-1315 of SEQ ID     NO: 8, amino acid 858-1268 of SEQ ID NO: 9, and amino acid 848-1251     of SEQ ID NO: 10. -   30. The multivalent Clostridial toxin according to 26, wherein the     Clostridial toxin binding domain comprises a BoNT/A H_(CC) binding     domain, a BoNT/B H_(CC) binding domain, a BoNT/C1 H_(CC) binding     domain, a BoNT/D H_(CC) binding domain, a BoNT/E H_(CC) binding     domain, a BoNT/F H_(CC) binding domain, a BoNT/G H_(CC) binding     domain, a TeNT H_(CC) binding domain, a BaNT H_(CC) binding domain,     and a BuNT H_(CC) binding domain. -   31. The multivalent Clostridial toxin according to 30, wherein the     Clostridial toxin binding domain comprises an amino acid sequence     selected from the group consisting of amino acids 874-1110 of SEQ ID     NO: 1, amino acids 861-1097 SEQ ID NO: 2, amino acids 869-1111 of     SEQ ID NO: 3, amino acids 865-1098 of SEQ ID NO: 4, amino acids     848-1085 of SEQ ID NO: 5, amino acids 867-1105 of SEQ ID NO: 6,     amino acids 866-1105 of SEQ ID NO: 7, amino acid 882-1127 of SEQ ID     NO: 8, amino acid 858-1094 of SEQ ID NO: 9, and amino acid 848-1085     of SEQ ID NO: 10. -   32. The multivalent Clostridial toxin according to 26, wherein the     Clostridial non-toxin associated protein β-trefoil domain is     selected from the group consisting of a BoNT/A HA-33 β-trefoil     domain, a BoNT/B HA-33 β-trefoil domain, a BoNT/C1 HA-33 β-trefoil     domain, a BoNT/D HA-33 β-trefoil domain, a BoNT/A HA-17 β-trefoil     domain, a BoNT/B HA-17 β-trefoil domain, a BoNT/C1 HA-17 β-trefoil     domain, a BoNT/D HA-17 β-trefoil domain, a BoNT/A NTNH β-trefoil     domain, a BoNT/B NTNH β-trefoil domain, a BoNT/C1 NTNH β-trefoil     domain, a BoNT/D NTNH β-trefoil domain, a BoNT/E NTNH β-trefoil     domain, a BoNT/F NTNH β-trefoil domain, AND a BoNT/G NTNH β-trefoil     domain. -   33. The multivalent Clostridial toxin according to 32, wherein the     BoNT/A HA-33 β-trefoil domain comprises amino acids 10-144 of SEQ ID     NO: 11, amino acids 151-293 of SEQ ID NO: 11, amino acids 10-144 or     amino acids 151-293 of SEQ ID NO: 12, amino acids 10-144 of SEQ ID     NO: 13, amino acids 151-293 of SEQ ID NO: 13, amino acids 10-146 of     SEQ ID NO: 14, amino acids 153-294 of SEQ ID NO: 14, amino acids     10-144 of SEQ ID NO: 15, or amino acids 151-279 of SEQ ID NO: 15. -   34. The multivalent Clostridial toxin according to 32, wherein the     BoNT/B HA-33 β-trefoil domain comprises amino acids 10-144 of SEQ ID     NO: 16, amino acids 151-292 of SEQ ID NO: 16, amino acids 10-146 of     SEQ ID NO: 17, or amino acids 153-291 of SEQ ID NO: 17. -   35. The multivalent Clostridial toxin according to 32, wherein the     BoNT/C1 HA-33 β-trefoil domain comprises amino acids 10-141 of SEQ     ID NO: 18, amino acids 148-285 of SEQ ID NO: 18, amino acids 10-141     of SEQ ID NO: 19, or amino acids 148-286 of SEQ ID NO: 19. -   36. The multivalent Clostridial toxin according to 32, wherein the     BoNT/D HA-33 β-trefoil domain comprises amino acids 10-141 of SEQ ID     NO: 20, or amino acids 148-286 of SEQ ID NO: 20. -   37. The multivalent Clostridial toxin according to 32, wherein the     BoNT/A HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID     NO: 21. -   38. The multivalent Clostridial toxin according to 32, wherein the     BoNT/B HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID     NO: 22. -   39. The multivalent Clostridial toxin according to 32, wherein the     BoNT/C1 HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID     NO: 23. -   40. The multivalent Clostridial toxin according to 32, wherein the     BoNT/D HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID     NO: 24. -   41. The multivalent Clostridial toxin according to 32, wherein the     BoNT/A NTNH β-trefoil domain comprises amino acids 1050-1194 of SEQ     ID NO: 25, amino acids 1050-1199 of SEQ ID NO: 26, or amino acids     1050-1194 of SEQ ID NO: 27. -   42. The multivalent Clostridial toxin according to 32, wherein the     BoNT/B NTNH β-trefoil domain comprises amino acids 1049-1198 of SEQ     ID NO: 28. -   43. The multivalent Clostridial toxin according to 32, wherein the     BoNT/C1 NTNH β-trefoil domain comprises amino acids 1049-1197 of SEQ     ID NO:29. -   44. The multivalent Clostridial toxin according to 32, wherein the     BoNT/D NTNH β-trefoil domain comprises amino acids 1049-1197 of SEQ     ID NO: 30. -   45. The multivalent Clostridial toxin according to 32, wherein the     BoNT/E NTNH β-trefoil domain comprises amino acids 1014-1163 of SEQ     ID NO: 31. -   46. The multivalent Clostridial toxin according to 32, wherein the     BoNT/F NTNH β-trefoil domain comprises amino acids 1016-1160 of SEQ     ID NO: 32, or amino acids 1017-1166 of SEQ ID NO: 33. -   47. The multivalent Clostridial toxin according to 32, wherein the     BoNT/G NTNH β-trefoil domain comprises amino acids 1050-1197 of SEQ     ID NO: 34. -   48. The multivalent Clostridial toxin according to 26, wherein the     FGF β-trefoil domain is selected from the group consisting of a     FGF-1 β-trefoil domain, a FGF-2 β-trefoil domain, a FGF-4 β-trefoil     domain, a FGF-8 β-trefoil domain, a FGF-9 β-trefoil domain, a FGF-17     β-trefoil domain and a FGF-18 β-trefoil domain. -   49. The multivalent Clostridial toxin according to 26, wherein the     FGF β-trefoil domain is selected from the group consisting of amino     acids 26-155 of SEQ ID NO: 35, amino acids 29-155 of SEQ ID NO: 36,     amino acids 83-206 of SEQ ID NO: 37, amino acids 43-172 of SEQ ID     NO: 38, amino acids 63-196 of SEQ ID NO: 39, amino acids 55-183 of     SEQ ID NO: 40 and amino acids 54-183 of SEQ ID NO: 41. -   50. The multivalent Clostridial toxin according either 1 or 13,     wherein the first binding domain comprises a binding domain selected     from the group consisting of a glucagon like hormone, a     neurohormone, a neuroregulatory cytokine, a neurotrophin, a growth     factor, and an axon guidance signaling molecule. -   51. The multivalent Clostridial toxin according to 50, wherein the     glucagon like hormone is selected from the group consisting of a     GRPP, a glucagon-like peptide-1 (GLP-1), a glucagon-like peptide-2     (GLP-2), a glucagon, an oxyntomodulin (OXY), pituitary adenylate     cyclase activating peptide (PACAP), GHRH, vasoactive intestinal     peptide-1 (VIP-1), vasoactive intestinal peptide-2 (VIP-2), gastric     inhibitory polypeptide (GIP), secretin, gastrin, GRP, Ghrelin (GHS),     glicentin, glicentin-related polypeptide (GRPP), and a     calcitonin-related peptidesvisceral gut peptide. -   52. The multivalent Clostridial toxin according to 50, wherein the     glucagon like hormone comprises amino acids 21-50 of SEQ ID NO: 42,     amino acids 53-81 of SEQ ID NO: 42, amino acids 53-89 of SEQ ID NO:     42, amino acids 98-124 of SEQ ID NO: 42, amino acids 146-178 of SEQ     ID NO: 42, amino acids 132-158 of SEQ ID NO: 43, amino acids 32-58,     of SEQ ID NO: 44, amino acids 32-75 of SEQ ID NO: 44, amino acids     81-107 of SEQ ID NO: 45, amino acids 125-151 of SEQ ID NO: 45, amino     acids 81-107 of SEQ ID NO: 46, amino acids 124-150 of SEQ ID NO: 46,     amino acids 52-78 of SEQ ID NO: 47, amino acids 52-93 of SEQ ID NO:     47, amino acids 28-54 of SEQ ID NO: 48, amino acids 76-92 of SEQ ID     NO: 49, amino acids 59-92 of SEQ ID NO: 49, amino acids 41-50 of SEQ     ID NO: 50, amino acids 24-50 of SEQ ID NO: 50, or amino acids 99-112     of SEQ ID NO: 51. -   53. The multivalent Clostridial toxin according to 50, wherein the     neurohormone is selected from the group consisting of     corticotropin-releasing hormone (CCRH) and parathyroid hormone     (PTH). -   54. The multivalent Clostridial toxin according to 50, wherein the     neurohormone comprises a amino acids 159-193 of SEQ ID NO: 52, amino     acids 154-194 of SEQ ID NO: 52, amino acids 35-70 of SEQ ID NO: 53     and amino acids 145-177 of SEQ ID NO: 53. -   55. The multivalent Clostridial toxin according to 50, wherein the     neuroregulatory cytokine is selected from the group consisting of     ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemia     inhibitory factor (LIF), an interleukin (IL), onostatin M (OSM),     cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),     neuroleukin (NL), VEGF, insulin-like growth factor-1 (IGF-1),     insulin-like growth factor-2 (IGF-2) and epidermal growth factor     (EGF). -   56. The multivalent Clostridial toxin according to 50, wherein the     neurohormone comprises SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,     SEQ ID NO: 57, SEQ ID NO: 58, amino acids 123-265 of SEQ ID NO: 59,     amino acids 21-153 of SEQ ID NO: 60, amino acids 57-210 of SEQ ID     NO: 61, amino acids 21-99 of SEQ ID NO: 62, amino acids 31-94 of SEQ     ID NO: 62, amino acids 37-173 of SEQ ID NO: 63, amino acids 19-178     of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, amino acids 52-109     of SEQ ID NO: 66, amino acids 49-118 of SEQ ID NO: 66, amino acids     31-84 of SEQ ID NO: 67, amino acids 25-180 of SEQ ID NO: 67 or SEQ     ID NO: 68. -   57. The multivalent Clostridial toxin according to 50, wherein the     neurotrophin is selected from the group consisting of nerve growth     factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3     (NT-3) and neurotrophin-4/5 (NT-4/5). -   58. The multivalent Clostridial toxin according to 50, wherein the     neurotrophin comprises amino acids 139-257 of SEQ ID NO: 69, amino     acids 133-240 of SEQ ID NO: 70, amino acids 129-247 of SEQ ID NO:     70, amino acids 144-249 of SEQ ID NO: 71, amino acids 19-257 of SEQ     ID NO: 71, amino acids 89-202 of SEQ ID NO: 72 and amino acids     81-210 of SEQ ID NO: 72. -   59. The multivalent Clostridial toxin according to 50, wherein the     growth factor is selected from the group consisting of glial cell     derived neurotrophic factor (GDNF), neurturin (NRTN), persephrin     (PSPN), artemin (ARTN), TGFβ1, TGFβ2, TGFβ3, TGFβ4, BMP2, BMP3,     BMP4, BMP5, BMP6, BMP7, BMP8, BMP10, GDF1, GDF2, GDF3, GDF5, GDF6,     GDF7, GDF8, GDF10, GDF11, GDF15, activin A, activin B, activin C,     activin E and inhibin A. -   60. The multivalent Clostridial toxin according to 50, wherein the     growth factor selected from the group consisting of amino acids     118-211 of SEQ ID NO: 73, amino acids 107-196 of SEQ ID NO: 74,     amino acids 96-197 of SEQ ID NO: 74, amino acids 66-155 of SEQ ID     NO: 75, amino acids 123-218 of SEQ ID NO: 76, amino acids 293-390 of     SEQ ID NO: 77, amino acids 317-414 of SEQ ID NO: 78, amino acids     315-412 of SEQ ID NO: 79, amino acids 276-373 of SEQ ID NO: 80,     amino acids 296-396 of SEQ ID NO: 81, amino acids 370-472 of SEQ ID     NO: 82, amino acids 309-409 of SEQ ID NO: 83, amino acids 353-454 of     SEQ ID NO: 84, amino acids 323-454 of SEQ ID NO: 84, amino acids     412-513 of SEQ ID NO: 85, amino acids 374-513 of SEQ ID NO: 85,     amino acids 330-431 of SEQ ID NO: 86, amino acids 293-431 of SEQ ID     NO: 86, amino acids 301-402 of SEQ ID NO: 87, amino acids 323-424 of     SEQ ID NO: 88, amino acids 267-372 of SEQ ID NO: 89, amino acids     327-429 of SEQ ID NO: 90, amino acids 264-364 of SEQ ID NO: 91,     amino acids 400-501 of SEQ ID NO: 92, amino acids 354-455 of SEQ ID     NO: 93, amino acids 352-450 of SEQ ID NO: 94, amino acids 281-375 of     SEQ ID NO: 95; amino acids 376-478 of SEQ ID NO: 96; amino acids     313-407 of SEQ ID NO: 97; amino acids 211-308 of SEQ ID NO: 98;     amino acids 321-426 of SEQ ID NO: 99; amino acids 303-406 of SEQ ID     NO: 100; amino acids 247-352 of SEQ ID NO: 101; amino acids 237-352     of SEQ ID NO: 101; amino acids 247-350 of SEQ ID NO: 102; amino     acids 262-366 of SEQ ID NO: 103; or amino acids 233-366 of SEQ ID     NO: 103. -   61. The multivalent Clostridial toxin according either 1 or 13,     wherein the first binding domain comprises a binding domain selected     from the group consisting of an opioid peptide, a melanocortin     peptide, a galanin peptide, a granin peptide, a tachykinin peptide,     a cholecystokinin peptide, a Neuropeptide Y related peptide, a kinin     peptide, a protease activated receptor (PAR) peptide, a somatostatin     peptide, a leukemia inhibitor factor peptide, and an interleukin-1     peptide. -   62. The multivalent Clostridial toxin according to 61, wherein the     opioid peptide is selected from the group consisting of an     enkephalin peptide, a BAM22 peptide, an endomorphin peptide, an     endorphin peptide, a dynorphin peptide, a nociceptin peptide and a     hemorphin peptide. -   63. The multivalent Clostridial toxin according to 62, wherein the     enkephalin peptide is selected from the group consisting of a     Leu-enkephalin, a Met-enkephalin, a Met-enkephalin MRGL and a     Met-enkephalin MRF -   64. The multivalent Clostridial toxin according to 62, wherein the     enkephalin peptide comprises SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID     NO: 106 or SEQ ID NO: 107. -   65. The multivalent Clostridial toxin according to 62, wherein the     BAM22 peptide is selected from the group consisting of a BAM22     peptide (1-12), a BAM22 peptide (6-22), a BAM22 peptide (8-22) or a     BAM22 peptide (1-22). -   66. The multivalent Clostridial toxin according to 62, wherein the     BAM22 peptide comprises amino acids 1-12 of SEQ ID NO: 108, amino     acids 6-22 of SEQ ID NO: 108, amino acids 8-22 of SEQ ID NO: 108,     amino acids 1-22 of SEQ ID NO: 108, amino acids 1-12 of SEQ ID NO:     109, amino acids 6-22 of SEQ ID NO: 109, amino acids 8-22 of SEQ ID     NO: 109, amino acids 1-22 of SEQ ID NO: 109, amino acids 1-12 of SEQ     ID NO: 110, amino acids 6-22 of SEQ ID NO: 110, amino acids 8-22 of     SEQ ID NO: 110, amino acids 1-22 of SEQ ID NO: 110, amino acids 1-12     of SEQ ID NO: 111, amino acids 6-22 of SEQ ID NO: 111, amino acids     8-22 of SEQ ID NO: 111, amino acids 1-22 of SEQ ID NO: 111, amino     acids 1-12 of SEQ ID NO: 112, amino acids 6-22 of SEQ ID NO: 112,     amino acids 8-22 of SEQ ID NO: 112, amino acids 1-22 of SEQ ID NO:     112, amino acids 1-12 of SEQ ID NO: 113, amino acids 6-22 of SEQ ID     NO: 113, amino acids 8-22 of SEQ ID NO: 113, or amino acids 1-22 of     SEQ ID NO: 113. -   67. The multivalent Clostridial toxin according to 62, wherein the     endomorphin peptide is selected from the group consisting of an     endomorphin-1 and an endomorphin-2. -   68. The multivalent Clostridial toxin according to 62, wherein the     endomorphin peptide comprises SEQ ID NO: 114, or SEQ ID NO: 115. -   69. The multivalent Clostridial toxin according to 62, wherein the     endorphin peptide is selected from the group consisting of an     endorphin-α, a neoendorphin-α, an endorphin-β, a neoendorphin-β or     an endorphin-γ. -   70. The multivalent Clostridial toxin according to 62, wherein the     altered targeting domain comprises an endorphin selected from the     group consisting of SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117,     SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120. -   71. The multivalent Clostridial toxin according to 62, wherein the     dynorphin peptide is selected from the group consisting of a     dynorphin A, a dynorphin B and a rimorphin. -   72. The multivalent Clostridial toxin according to 62, wherein the     dynorphin peptide comprises SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID     NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:     127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131,     SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ     ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID     NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:     144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148,     SEQ ID NO: 149, SEQ ID NO: 150, or SEQ ID NO: 151. -   73. The multivalent Clostridial toxin according to 62, wherein the     nociceptin peptide selected from the group consisting of a     nociceptin RK, a nociceptin, a neuropeptide 1, a neuropeptide 2 or a     neuropeptide 3. -   74. The multivalent Clostridial toxin according to 62, wherein the     nociceptin peptide comprises SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID     NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:     158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161. -   75. The multivalent Clostridial toxin according to 61, wherein the     melanocortin peptide is selected from the group consisting of an     α-melanocyte stimulating hormones (α-MSH), α-melanocyte stimulating     hormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH), an     adrenocorticotropin (ACTH), a Corticotropin-like intermediary     peptide (CLIP), a β-lipotropin (β-LPH) and a γ-lipotropin (γ-LPH). -   76. The multivalent Clostridial toxin according to 61, wherein the     melanocortin peptide is selected from the group consisting SEQ ID     NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:     166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170,     or SEQ ID NO: 171. -   77. The multivalent Clostridial toxin according to 61, wherein the     galanin peptide is selected from the group consisting of a galanin     and a galanin message-associated peptide (GMAP). -   78. The multivalent Clostridial toxin according to 61, wherein the     galanin peptide comprises SEQ ID NO: 172, or SEQ ID NO: 173. -   79. The multivalent Clostridial toxin according to 61, wherein the     grainin peptide is selected from the group consisting of a     chromogranin A peptide, a chromogranin B peptide and a chromogranin     C peptide. -   80. The multivalent Clostridial toxin according to 79, wherein the     chromogranin A peptide is selected from the group consisting of a     β-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, a     catestatin, a parastatin and a GE-25. -   81. The modified Clostridial toxin according to 79, wherein the     chromogranin A peptide comprises SEQ ID NO: 174, SEQ ID NO: 175, SEQ     ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID     NO: 180, or SEQ ID NO: 181. -   82. The multivalent Clostridial toxin according to 79, wherein the     chromogranin B peptide is selected from the group consisting of a     GAWK peptide, an adrenomedullary peptide and a secretolytin. -   83. The multivalent Clostridial toxin according to 79, wherein the     chromogranin B peptide comprises SEQ ID NO: 182, SEQ ID NO: 183, SEQ     ID NO: 184, SEQ ID NO: 185, or SEQ ID NO: 186 -   84. The multivalent Clostridial toxin according to 79, wherein the     chromogranin C peptide is selected from the group consisting of a     secretoneurin, a EM66 and a manserin. -   85. The multivalent Clostridial toxin according to 79, wherein the     chromogranin C peptide comprises SEQ ID NO: 187. -   86. The multivalent Clostridial toxin according to 61, wherein the     tachykinin peptide is selected from the group consisting of a     Substance P, a neuropeptide K (NPK), a neuropeptide gamma, a     neurokinin A, a neurokinin B, a hemokinin and a endokinin. -   87. The multivalent Clostridial toxin according to 61, wherein the     tachykinin peptide comprises SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID     NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO:     194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198     and SEQ ID NO: 199. -   88. The multivalent Clostridial toxin according to 61, wherein the     cholecystokinin peptide is selected from the group consisting of a     cholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, a     cholecystokinin 12 and a cholecystokinin 8. -   89. The multivalent Clostridial toxin according to 88, wherein the     cholecystokinin 58 is selected from the group consisting of SEQ ID     NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:     204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,     SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ     ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 215. -   90. The multivalent Clostridial toxin according to 88, wherein the     cholecystokinin 39 is selected from the group consisting of amino     acids 20-58 of SEQ ID NO: 200, amino acids 20-58 of SEQ ID NO: 201,     amino acids 20-58 of SEQ ID NO: 202, amino acids 20-58 of SEQ ID NO:     203, amino acids 20-58 of SEQ ID NO: 204, amino acids 20-58 of SEQ     ID NO: 205, amino acids 20-58 of SEQ ID NO: 207, amino acids 20-58     of SEQ ID NO: 208, amino acids 20-58 of SEQ ID NO: 209, amino acids     20-58 of SEQ ID NO: 210, amino acids 20-58 of SEQ ID NO: 211, amino     acids 20-58 of SEQ ID NO: 212, amino acids 20-58 of SEQ ID NO: 213,     amino acids 20-58 of SEQ ID NO: 214, or amino acids 20-58 of SEQ ID     NO: 215. -   91. The multivalent Clostridial toxin according to 88, wherein the     cholecystokinin 33 is selected from the group consisting of amino     acids 26-58 of SEQ ID NO: 200, amino acids 26-58 of SEQ ID NO: 201,     amino acids 26-58 of SEQ ID NO: 202, amino acids 26-58 of SEQ ID NO:     203, amino acids 26-58 of SEQ ID NO: 204, amino acids 26-58 of SEQ     ID NO: 205, amino acids 26-58 of SEQ ID NO: 207, amino acids 26-58     of SEQ ID NO: 208, amino acids 26-58 of SEQ ID NO: 209, amino acids     26-58 of SEQ ID NO: 210, amino acids 26-58 of SEQ ID NO: 211, amino     acids 26-58 of SEQ ID NO: 212, amino acids 26-58 of SEQ ID NO: 213,     amino acids 26-58 of SEQ ID NO: 214 or amino acids 26-58 of SEQ ID     NO: 215. -   92. The multivalent Clostridial toxin according to 88, wherein the     cholecystokinin 12 is selected from the group consisting of amino     acids 47-58 of SEQ ID NO: 200, amino acids 47-58 of SEQ ID NO: 210     or amino acids 47-58 of SEQ ID NO: 214. -   93. The multivalent Clostridial toxin according to 88, wherein the     cholecystokinin 8 is amino acids 51-58 of SEQ ID NO: 200. -   94. The multivalent Clostridial toxin according to 61, wherein the     Neuropeptide Y related peptide is selected from the group consisting     of a Neuropeptide Y (NPY), a Peptide YY (PYY), a Pancreatic peptide     (PP) and a Pancreatic icosapeptide (PIP). -   95. The multivalent Clostridial toxin according to 61, wherein the     Neuropeptide Y related peptide comprises SEQ ID NO: 216, SEQ ID NO:     217, SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220. -   96. The multivalent Clostridial toxin according to 61, wherein the     kinin peptide is selected from the group consisting of a bradykinin,     a kallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin. -   97. The multivalent Clostridial toxin according to 61, wherein the     kinin peptide comprises SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:     229, or SEQ ID NO: 230. -   98. The multivalent Clostridial toxin according to 61, wherein the     PAR peptide is selected from the group consisting of a PAR1 peptide,     a PAR2 peptide, a PAR3 peptide and a PAR4 peptide. -   99. The modified Clostridial toxin according to 61, wherein the PAR     peptide comprises amino acids 42-47, amino acids 42-55, amino acids     29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40,     amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID     NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or     amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids     48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234. -   100. The multivalent Clostridial toxin according either 1 or 13,     wherein the first binding domain comprises a binding domain selected     from the group consisting of a PTD. -   101. The multivalent Clostridial toxin according to 100, wherein the     PTD is selected from the group consisting of a herpes simplex virus     type 1 VP22 protein translocating sequence, a SV-40 virus large T     translocating sequence, a TAT translocating sequence, an adenovirus     translocating sequence, a synthetic integrin binding domain     translocating sequence, a Kaposi fibroblast growth factor membrane     translocating sequence, a nuclear localization signal, a Transportan     translocating sequence, a ciliary neurotrophic factor translocating     sequence, a caveolin, an interleukin 1-β translocating sequence, a     thioredoxin translocating sequence, a fibroblast growth factor-1     translocating sequence, a fibroblast growth factor-2 translocating     sequence, an integrin β1 translocating sequence, an integrin β3     translocating sequence, a lactoferrin translocating sequence, a     homeodomain translocating sequence, like, a penetratin translocating     sequence, an Engrailed-1 translocating sequence, an Engrailed-2     translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4     translocating sequence, and a Hoxc-8 translocating sequence. -   102. The multivalent Clostridial toxin according to 100, wherein the     PTD comprises SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID     NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO:     242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246,     SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ     ID NO: 251, SEQ ID NO: 252, or SEQ ID NO: 253. -   103. A polynucleotide molecule encoding a multivalent Clostridial     toxin according to any one of Claims 1-102. -   104. The polynucleotide molecule according to Claims 103, wherein     the polynucleotide molecule is an expression construct. -   105. A method of producing a multivalent Clostridial toxin     comprising the step of expressing in a cell a polynucleotide     according to 52 -   106. A method of producing a multivalent Clostridial toxin     comprising the steps of     -   a) introducing in a cell an expression construct comprising a         polynucleotide according to 52; and     -   b) expressing the expression construct in the cell.

EXAMPLES Example 1 Construction of a Multivalent Clostridial Neurotoxin Comprising Two Clostridial Toxin Binding Domains

This example illustrates how to make a multivalent Clostridial toxin comprising two modified Clostridial toxin binding domains with enhanced binding activity using site-directed mutagenesis.

A polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) and further comprising a polynucleotide sequence comprising a repeat of amino acids 874-1296 of the binding domain of BoNT/A at the amino terminus is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.). The basic strategy is set forth in FIG. 5A. Oligonucleotides of 20 to 50 bases in length are synthesized using standard phosphoramidite synthesis. These oligonucleotides are hybridized into double stranded duplexes that are ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule is cloned using standard molecular biology methods into a pUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/multivalent Clostridial toxin(AA)1. The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

If desired, an expression-optimized polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) can be synthesized in order to improve expression in an Escherichia coli strain. The polynucleotide molecule encoding the BoNT/A can be modified to 1) contain synonymous codons typically present in native polynucleotide molecules of an Escherichia coli strain; 2) contain a G+C content that more closely matches the average G+C content of native polynucleotide molecules found in an Escherichia coli strain; 3) reduce polymononucleotide regions found within the polynucleotide molecule; and/or 4) eliminate internal regulatory or structural sites found within the polynucleotide molecule, see, e.g., Lance E. Steward et al. Optimizing Expression of Active Botulinum Toxin Type E, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al. Optimizing Expression of Active Botulinum Toxin Type A, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization is complete, oligonucleotides of 20 to 50 bases in length are synthesized using standard phosphoramidite synthesis. These oligonucleotides are hybridized into double stranded duplexes that are ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule is cloned using standard molecular biology methods into a pUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/multivalent Clostridial toxin1/(AA)1:ECopt. The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.). Is so desired, optimization to a different organism, such as, e.g., a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructs comprising a polynucleotide molecule encoding BoNT/B of SEQ ID NO: 2; a polynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; a polynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; a polynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; a polynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; a polynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; a polynucleotide molecule encoding TeNT of SEQ ID NO: 8; a polynucleotide molecule encoding BaNT of SEQ ID NO: 9; a polynucleotide molecule encoding BuNT of SEQ ID NO: 10; wherein the additional binding domain can be selected from, e.g., any of the H_(C) or H_(CC) binding domains listed in Table 1. In addition, one skilled in the art can modify Clostridial toxins, such as, e.g., to include an exogenous protease cleavage site within the di-chain loop region, or flexible spacer regions. Likewise, a similar cloning strategy can be used to make other domain orientations as, e.g., as set forth in FIG. 4A, 4B, 4C, 4D, 5B, 5C, 5D, 6A or 6B.

To construct pET29/multivalent Clostridial toxin/A(AA)1, a pUCBHB1/multivalent Clostridial toxin(AA)1 construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame of SEQ ID NO: 1 (with the added nucleotide sequence encoding the N terminal Binding 1 site) encoding the multivalent Clostridial toxin(AA)1; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/multivalent Clostridial toxin/A(AA)1. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yields a pET29 expression construct comprising the polynucleotide molecule encoding the BoNT/A of SEQ ID NO: 1 operably-linked to an additional N-terminal binding site comprising the native binding region of BoNT/A.

A similar cloning strategy is used to make pET29 expression constructs comprising polynucleotide molecule encoding the multivalent Clostridial toxins discussed above comprising BoNT/B of SEQ ID NO: 2; a polynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; a polynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; a polynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; a polynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; a polynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; a polynucleotide molecule encoding TeNT of SEQ ID NO: 8, a polynucleotide molecule encoding BaNT of SEQ ID NO: 9 and a polynucleotide molecule encoding BuNT of SEQ ID NO: 10, each with an additional binding site.

To construct a multivalent Clostridial toxin comprising one or more modified binding domain with enhanced binding activity, specific amino acids influencing binding activity will be changed. For example, it is already known that amino acids Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270, Gly 1279 and Trp 1282 of SEQ ID NO: 1 (within the binding site) are important for function. To determine which amino acid substitutions could enhance the binding activity of a BoNT/A binding domain, computational protein design algorithms will generate novel binding domains with optimized properties. The crystal structure of a BoNT/A binding domain will be used as the starting template for computational calculations. Potential amino acid candidates will be identified using a combined output from Protein Design Automation® (PDA®) and Sequence Prediction Algorithm™ (SPA™) calculations. For PDA calculations, the conformations of amino acids at variable positions will be represented as a set of backbone-independent side chain rotamers derived from the rotamer library. The energies of all possible combinations of the considered amino acids at the chosen variable positions will be calculated using a force field containing terms describing van der Waals, solvation, electrostatic, and hydrogen bond interactions. The optimal (ground state) sequence will be determined using a Dead End Elimination (DEE) algorithm, and a Monte Carlo (MC) algorithm will be used to evaluate the energies of similar sequences around the predicted ground state. SPA calculations utilize a genetic algorithm to screen for low energy sequences, with energies being calculated during each round of “evolution” for those sequences being sampled. The conformations of amino acids will be represented as a set of side chain rotamers derived from a backbone-independent rotamer library using a flexible rotamer model. SPA calculations will generate sequences which will be subsequently clustered computationally into groups of similar sequences using a nearest neighbor single linkage hierarchical clustering algorithm. Critical contact amino acids will be fixed in both sequence and conformation and calculations will be carried out to evaluate single and combinatorial substitutions at variable amino acids. All amino acids in contact with these residues will be floated, that is the amino acid conformation but not the amino acid identity will be allowed to vary to allow for conformational adjustments. Final experimental substitutions will be chosen based on their predicted energies relative to the naturally occurring BoNT/A binding domain and their occupancy, that is the number times the substitution occurred in the set of 1000 MC or genetic algorithm sequences. Two sets of design calculations will be carried out using Rosetta to identify substitutions predicted to stabilize the BoNT/A binding domain. In the first round, only single amino acid substitutions will be modeled. In a second round, interface amino acids will be allowed to change to all 20 naturally occurring amino acids including the native amino acid type, but excluding cysteine, simultaneously. In each case, amino acid side chains contacting the substituted amino acid side chains will be repacked (allowing all rotamers of the native amino acid type). Sequences and conformations with low energies will be selected using a Monte-Carlo simulated annealing procedure. All resulting protein complex models will be rescored by computing a predicted binding energy. Final sequences are selected for the lowest binding energy.

To use this information to generate one or more modified Clostridial neurotoxin binding domain, candidate amino acids identified as described above will be changed using site-directed in vitro mutagenesis. A 50 μL reaction will be assembled using pET29/multivalent Clostridial toxin/A(AA)1 as a template, sense and antisense oligonucleotides encoding the desired amino acid change identified above, and reagents included with the QuickChange® II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.). The polymerase chain reaction (PCR) mix will contain 5 μL of 10× Buffer, 1 μL of deoxyribonucleotides (dNTPs), 1 μL of PfuUltra™ High Fidelity DNA polymerase (2.5 units/μL), 125 ng of each primer, 100 ng of template DNA, and nuclease-free water to a final volume of 50 μL. The thermocycler conditions will be: one cycle of 95° C. for 60 seconds; 16 cycles of 95° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for 10 minutes; one cycle of 72° C. for 5 minutes; and 4° C. to hold. Following thermocycling, 1 μL of DpnI restriction enzyme (Stratagene, La Jolla, Calif.) will be added to the reaction and will be incubated for 1 hour at 37° C. to digest the template DNA. The reaction will be purified by QIAquick kit (QIAGEN, Inc., Valencia, Calif.) and will be analysis by agarose gel electrophoresis to determine that the reaction produced full-length plasmid. The mutagenesis products will be transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of Ampicillin, and will be placed in a 37° C. incubator for overnight growth. Candidate mutagenesis constructs will be isolated as Ampicillin resistant colonies and will be analyzed using an alkaline lysis plasmid mini-preparation procedure to isolate the expression construct and restriction endonuclease digests to determine the presence of the insert. The incorporation of the point mutation will be determined by sequence analysis of candidate plasmid constructs.

To test the binding activity of multivalent Clostridial toxins comprising binding domains derived from BoNT/A, the soluble portion of FGFR3 will be expressed recombinantly for use in surface plasmon resonance (SPR) binding assays, e.g., Biacore® (Biacore Inc., Piscataway, N.J.). The soluble portion of FGFR3 will be expressed as a fusion to streptavidin and the receptor will then be immobilized on an appropriate sensor chip. Utilizing a Biacore® instrument, changes in local refractive index as a result of receptor binding will be measured as a change in the SPR angle. The rates of change in the SPR angle will then be analyzed to determine association rate (K_(on)), dissociation rate (K_(off)) and the dissociation equilibrium constant (K_(D)=K_(off)/K_(on)). Multivalent Clostridial neurotoxin derivatives comprising binding domains derived from BoNT/A exhibit either an increased association rate, a decreased dissociation rate, both an increased association rate and a decreased dissociation rate, or a decreased dissociation equilibrium constant relative to the measurements obtained from the naturally occurring BoNT/A from which the multivalent Clostridial toxin is derived.

The same methods and rationale may be used to make and test the affinity of any multivalent Clostridial toxin comprising additional binding domain derived from a Clostridial toxin. Generally, but not exclusively, a multivalent Clostridial toxin is tested relative to the Clostridial toxin with which it shares the greatest homology, particularly in the binding domains.

Example 2 Construction of a Multivalent Clostridial Neurotoxin Comprising a Non-Toxin Associated Protein

A polynucleotide molecule encoding BoNT/A-Nterm33/A is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-Nterm33/A is a BoNT/A modified to replace amino acids in the second binding domain (the N-terminal binding region of the multivalent Clostridial toxin described in Example 1) corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with amino acids 151 to 293 of SEQ ID NO: 9, a HA-33 β-trefoil domain from a Clostridial botulinum serotype A strain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-Nterm33/A can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructs for multivalent Clostridial toxin BoNT/B-Nterm33/A, a multivalent Clostridial toxin based on BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; multivalent Clostridial toxin BoNT/C1-Nterm33/A, a multivalent Clostridial toxin based on BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; multivalent Clostridial toxin BoNT/D-Nterm33/A, a multivalent Clostridial toxin based on BoNT/D where amino acids 1099-1276 of SEQ ID NO: 4 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; multivalent Clostridial toxin BoNT/E-Nterm33/A, a multivalent Clostridial toxin based on BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; multivalent Clostridial toxin BoNT/F-Nterm33/A, a multivalent Clostridial toxin based on BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; multivalent Clostridial toxin BoNT/G-Nterm33/A, a multivalent Clostridial toxin based on BoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; and multivalent Clostridial toxin TeNT-Nterm33/A, a modified TeNT where amino acids 1128-1315 of SEQ ID NO: 8 are replaced with amino acids 151 to 293 of SEQ ID NO: 11.

Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a non-toxin associated protein β-trefoil domain comprising amino acids 10-144 of SEQ ID NO: 11; amino acids 10-144 of SEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-146 of SEQ ID NO: 14; amino acids 10-144 of SEQ ID NO: 15; amino acids 10-144 of SEQ ID NO: 16; amino acids 10-146 of SEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; amino acids 10-141 of SEQ ID NO: 19; amino acids 10-141 of SEQ ID NO: 20; amino acids 151-293 of SEQ ID NO: 12; amino acids 151-293 of SEQ ID NO: 13; amino acids 153-294 of SEQ ID NO: 14; amino acids 151-279 of SEQ ID NO: 15; amino acids 151-292 of SEQ ID NO: 16; amino acids 153-291 of SEQ ID NO: 17; amino acids 148-285 of SEQ ID NO: 18; amino acids 148-286 of SEQ ID NO: 19; amino acids 148-286 of SEQ ID NO: 20; amino acids 9-146 of SEQ ID NO: 21; amino acids 9-146 of SEQ ID NO: 22; amino acids 9-146 of SEQ ID NO: 23; amino acids 9-146 of SEQ ID NO: 24; amino acids 1050-1194 of SEQ ID NO: 25; amino acids 1050-1199 of SEQ ID NO: 26; amino acids 1050-1194 of SEQ ID NO: 27; amino acids 1049-1198 of SEQ ID NO: 28; amino acids 1049-1197 of SEQ ID NO: 29; amino acids 1049-1197 of SEQ ID NO: 30; amino acids 1014-1163 of SEQ ID NO: 31; amino acids 1016-1160 of SEQ ID NO: 32; amino acids 1017-1166 of SEQ ID NO: 33; and amino acids 1050-1199 of SEQ ID NO: 34.

To construct pET29/multivalent Clostridial toxin/BoNT/A-Nterm33/A, a pUCBHB1/BoNT/A-33/A construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding the BoNT/A-Nterm33/A; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/BoNT/A-Nterm33/A. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding a multivalent Clostridial toxin BoNT/A-Nterm33/A operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding for BoNT/B-33/A, BoNT/C1-33/A, BoNT/D-33/A, BoNT/E-33/A, BoNT/F-33/A, BoNT/G-33/A, TeNT-33/A, as well as the modified Clostridial toxin indicated above comprising amino acids 10-144 of SEQ ID NO: 11; amino acids 10-144 of SEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-146 of SEQ ID NO: 14; amino acids 10-144 of SEQ ID NO: 15; amino acids 10-144 of SEQ ID NO: 16; amino acids 10-146 of SEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; amino acids 10-141 of SEQ ID NO: 19; amino acids 10-141 of SEQ ID NO: 20; amino acids 151-293 of SEQ ID NO: 12; amino acids 151-293 of SEQ ID NO: 13; amino acids 153-294 of SEQ ID NO: 14; amino acids 151-279 of SEQ ID NO: 15; amino acids 151-292 of SEQ ID NO: 16; amino acids 153-291 of SEQ ID NO: 17; amino acids 148-285 of SEQ ID NO: 18; amino acids 148-286 of SEQ ID NO: 19; amino acids 148-286 of SEQ ID NO: 20; amino acids 9-146 of SEQ ID NO: 21; amino acids 9-146 of SEQ ID NO: 22; amino acids 9-146 of SEQ ID NO: 23; amino acids 9-146 of SEQ ID NO: 24; amino acids 1050-1194 of SEQ ID NO: 25; amino acids 1050-1199 of SEQ ID NO: 26; amino acids 1050-1194 of SEQ ID NO: 27; amino acids 1049-1198 of SEQ ID NO: 28; amino acids 1049-1197 of SEQ ID NO: 29; amino acids 1049-1197 of SEQ ID NO: 30; amino acids 1014-1163 of SEQ ID NO: 31; amino acids 1016-1160 of SEQ ID NO: 32; amino acids 1017-1166 of SEQ ID NO: 33; and amino acids 1050-1199 of SEQ ID NO: 34.

Example 3 Construction of a Multivalent Clostridial Toxin Comprising an FGF

A polynucleotide molecule encoding BoNT/A-F18 is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-F18 is a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with amino acids 54 to 183 of SEQ ID NO: 39, a FGF-18 β-trefoil domain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-F18 can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A polynucleotide molecule encoding BoNT/A-NtermF18 is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-NtermF18 is a BoNT/A modified to replace amino acids in the second binding domain (the N-terminal binding region of the multivalent Clostridial toxin described in Example 1) corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with amino acids 54 to 183 of SEQ ID NO: 41, a FGF-18 β-trefoil domain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-NtermF18 can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructs for multivalent Clostridial toxin BoNT/B-F18, a modified BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; multivalent Clostridial toxin BoNT/C1-F18, a modified BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; BoNT/D-F18, multivalent Clostridial toxin BoNT/D where amino acids 1099-1276 of SEQ ID NO: 4 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; BoNT/E-F18, multivalent Clostridial toxin BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; BoNT/F-F18, multivalent Clostridial toxin BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; BoNT/G-F18, multivalent Clostridial toxin BoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; multivalent Clostridial toxin TeNT-F18, TeNT where amino acids 1128-1315 of SEQ ID NO: 8 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; multivalent Clostridial toxin BaNT-F18, BaNT where amino acids 1095-1268 of SEQ ID NO: 9 are replaced with amino acids 54 to 183 of SEQ ID NO: 41; multivalent Clostridial toxin BuNT-F18, BuNT where amino acids 1086-1251 of SEQ ID NO: 10 are replaced with amino acids 54 to 183 of SEQ ID NO: 41. Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a FGF β-trefoil domain comprising amino acids 26-155 of SEQ ID NO: 35; amino acids 29-155 of SEQ ID NO: 36; amino acids 83-206 of SEQ ID NO: 37; amino acids 43-172 of SEQ ID NO: 38; amino acids 63-196 of SEQ ID NO: 39; and amino acids 55-183 of SEQ ID NO: 40.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermF18, a pUCBHB1/BoNT/A-F18 construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding BoNT/A-NtermF18; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/BoNT/A-NtermF18. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding the multivalent Clostridial toxin BoNT/A-NtermF18 operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding multivalent Clostridial toxins BoNT/B-F18, BoNT/C1-F18, BoNT/D-F18, BoNT/E-F18, BoNT/F-F18, BoNT/G-F18, TeNT-F18, as well as multivalent Clostridial toxin indicated above comprising amino acids 26-155 of SEQ ID NO: 35; amino acids 29-155 of SEQ ID NO: 36; amino acids 83-206 of SEQ ID NO: 37; amino acids 43-172 of SEQ ID NO: 38; amino acids 63-196 of SEQ ID NO: 39; and amino acids 55-183 of SEQ ID NO: 40.

Example 4 Construction of a Multivalent Clostridial Neurotoxin Comprising a Binding Domain

A polynucleotide molecule encoding BoNT/A-NtermGRPP is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-NtermGRPP is a BoNT/A modified to replace amino acids in the second binding domain (the N-terminal binding region of the multivalent Clostridial toxin described in Example 1) corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with amino acids 21 to 50 of SEQ ID NO: 42, a GRPP binding domain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-NtermGRPP can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructs for multivalent Clostridial toxin BoNT/B-GRPP, a modified BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; multivalent Clostridial toxin BoNT/C1-GRPP, a modified BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; BoNT/D-GRPP, multivalent Clostridial toxin BoNT/D where amino acids 1099-1276 of SEQ ID NO: 4 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; BoNT/E-GRPP, multivalent Clostridial toxin BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; BoNT/F-GRPP, multivalent Clostridial toxin BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; BoNT/G-GRPP, multivalent Clostridial toxin BoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; multivalent Clostridial toxin TeNT-GRPP, TeNT where amino acids 1128-1315 of SEQ ID NO: 8 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; multivalent Clostridial toxin BaNT-GRPP, BaNT where amino acids 1095-1268 of SEQ ID NO: 9 are replaced with amino acids 21 to 50 of SEQ ID NO: 42; multivalent Clostridial toxin BuNT-GRPP, BuNT where amino acids 1086-1251 of SEQ ID NO: 10 are replaced with amino acids 21 to 50 of SEQ ID NO: 42.

Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a binding domain comprising, e.g., amino acids 53-81 of SEQ ID NO: 42; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 42; amino acids 146-178 of SEQ ID NO: 42; amino acids 132-158 of SEQ ID NO: 43; amino acids 32-58 of SEQ ID NO: 44; amino acids 32-75 of SEQ ID NO: 44; amino acids 81-107 of SEQ ID NO: 45; amino acids 125-151 of SEQ ID NO: 45; amino acids 81-107 of SEQ ID NO: 46; amino acids 124-150 of SEQ ID NO: 46; amino acids 52-78 of SEQ ID NO: 47; amino acids 52-93 of SEQ ID NO: 47; amino acids 28-54 of SEQ ID NO: 48; amino acids 76-92 of SEQ ID NO: 49; amino acids 59-92 of SEQ ID NO: 49; amino acids 41-50 of SEQ ID NO: 50; amino acids 24-50 of SEQ ID NO: 50; amino acids 99-112 of SEQ ID NO: 51; amino acids 159-193 of SEQ ID NO: 52; amino acids 154-194 of SEQ ID NO: 52; amino acids 35-70 of SEQ ID NO: 53; amino acids 145-177 of SEQ ID NO: 53; amino acids 1-200 of SEQ ID NO: 54; amino acids 1-150 of SEQ ID NO: 55; amino acids 1-202 of SEQ ID NO: 56; amino acids 1-201 of SEQ ID NO: 57; amino acids 1-225 of SEQ ID NO: 58; amino acids 123-265 of SEQ ID NO: 59; amino acids 21-153 of SEQ ID NO: 60; amino acids 57-210 of SEQ ID NO: 61; amino acids 21-99 of SEQ ID NO: 62; amino acids 31-94 of SEQ ID NO: 62; amino acids 19-178 of SEQ ID NO: 63; amino acids 1-558 of SEQ ID NO: 64; amino acids 1-371 of SEQ ID NO: 65; amino acids 49-118 of SEQ ID NO: 66; amino acids 25-180 of SEQ ID NO: 67; amino acids 1-54 of SEQ ID NO: 68; amino acids 139-257 of SEQ ID NO: 69; amino acids 129-247 of SEQ ID NO: 70; amino acids 19-257 of SEQ ID NO: 71; amino acids 81-210 of SEQ ID NO: 72; amino acids 118-211 of SEQ ID NO: 73; amino acids 107-196 of SEQ ID NO: 74; amino acids 96-197 of SEQ ID NO: 74; amino acids 66-155 of SEQ ID NO: 75; amino acids 123-218 of SEQ ID NO: 76; amino acids 293-390 of SEQ ID NO: 77; amino acids 317-414 of SEQ ID NO: 78; amino acids 315-412 of SEQ ID NO: 79; amino acids 276-373 of SEQ ID NO: 80; amino acids 296-396 of SEQ ID NO: 81; amino acids 370-472 of SEQ ID NO: 82; amino acids 309-409 of SEQ ID NO: 83; amino acids 323-454 of SEQ ID NO: 84; amino acids 412-513 of SEQ ID NO: 85; amino acids 374-513 of SEQ ID NO: 85; amino acids 330-431 of SEQ ID NO: 86; amino acids 293-431 of SEQ ID NO: 86; amino acids 301-402 of SEQ ID NO: 87; amino acids 323-424 of SEQ ID NO: 88; amino acids 267-372 of SEQ ID NO: 89; amino acids 327-429 of SEQ ID NO: 90; amino acids 264-364 of SEQ ID NO: 91; amino acids 400-501 of SEQ ID NO: 92; amino acids 354-455 of SEQ ID NO: 93; amino acids 352-450 of SEQ ID NO: 94; amino acids 281-375 of SEQ ID NO: 95; amino acids 376-478 of SEQ ID NO: 96; amino acids 313-407 of SEQ ID NO: 97; amino acids 211-308 of SEQ ID NO: 98; amino acids 321-426 of SEQ ID NO: 99; amino acids 303-406 of SEQ ID NO: 100; amino acids 247-352 of SEQ ID NO: 101; amino acids 237-352 of SEQ ID NO: 101; amino acids 247-350 of SEQ ID NO: 102; amino acids 262-366 of SEQ ID NO: 103; or amino acids 233-366 of SEQ ID NO: 103.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermGRPP, a pUCBHB1/BoNT/A-GRPP construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding the BoNT/A-NtermGRPP; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/BoNT/A-NtermGRPP. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding the multivalent Clostridial toxin BoNT/A-NtermGRPP operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding multivalent Clostridial toxins BoNT/B-GRPP, BoNT/C1-GRPP, BoNT/D-GRPP, BoNT/E-GRPP, BoNT/F-GRPP, BoNT/G-GRPP, TeNT-GRPP, BaNT-GRPP, BuNT-GRPP, as well as multivalent Clostridial toxin indicated above comprising, e.g., amino acids 53-81 of SEQ ID NO: 42; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 42; amino acids 146-178 of SEQ ID NO: 42; amino acids 132-158 of SEQ ID NO: 43; amino acids 32-58 of SEQ ID NO: 44; amino acids 32-75 of SEQ ID NO: 44; amino acids 81-107 of SEQ ID NO: 45; amino acids 125-151 of SEQ ID NO: 45; amino acids 81-107 of SEQ ID NO: 46; amino acids 124-150 of SEQ ID NO: 46; amino acids 52-78 of SEQ ID NO: 47; amino acids 52-93 of SEQ ID NO: 47; amino acids 28-54 of SEQ ID NO: 48; amino acids 76-92 of SEQ ID NO: 49; amino acids 59-92 of SEQ ID NO: 49; amino acids 41-50 of SEQ ID NO: 50; amino acids 24-50 of SEQ ID NO: 50; amino acids 99-112 of SEQ ID NO: 51; amino acids 159-193 of SEQ ID NO: 52; amino acids 154-194 of SEQ ID NO: 52; amino acids 35-70 of SEQ ID NO: 53; amino acids 145-177 of SEQ ID NO: 53; amino acids 1-200 of SEQ ID NO: 54; amino acids 1-150 of SEQ ID NO: 55; amino acids 1-202 of SEQ ID NO: 56; amino acids 1-201 of SEQ ID NO: 57; amino acids 1-225 of SEQ ID NO: 58; amino acids 123-265 of SEQ ID NO: 59; amino acids 21-153 of SEQ ID NO: 60; amino acids 57-210 of SEQ ID NO: 61; amino acids 21-99 of SEQ ID NO: 62; amino acids 31-94 of SEQ ID NO: 62; amino acids 19-178 of SEQ ID NO: 63; amino acids 1-558 of SEQ ID NO: 64; amino acids 1-371 of SEQ ID NO: 65; amino acids 49-118 of SEQ ID NO: 66; amino acids 25-180 of SEQ ID NO: 67; amino acids 1-54 of SEQ ID NO: 68; amino acids 139-257 of SEQ ID NO: 69; amino acids 129-247 of SEQ ID NO: 70; amino acids 19-257 of SEQ ID NO: 71; amino acids 81-210 of SEQ ID NO: 72; amino acids 118-211 of SEQ ID NO: 73; amino acids 107-196 of SEQ ID NO: 74; amino acids 96-197 of SEQ ID NO: 74; amino acids 66-155 of SEQ ID NO: 75; amino acids 123-218 of SEQ ID NO: 76; amino acids 293-390 of SEQ ID NO: 77; amino acids 317-414 of SEQ ID NO: 78; amino acids 315-412 of SEQ ID NO: 79; amino acids 276-373 of SEQ ID NO: 80; amino acids 296-396 of SEQ ID NO: 81; amino acids 370-472 of SEQ ID NO: 82; amino acids 309-409 of SEQ ID NO: 83; amino acids 323-454 of SEQ ID NO: 84; amino acids 412-513 of SEQ ID NO: 85; amino acids 374-513 of SEQ ID NO: 85; amino acids 330-431 of SEQ ID NO: 86; amino acids 293-431 of SEQ ID NO: 86; amino acids 301-402 of SEQ ID NO: 87; amino acids 323-424 of SEQ ID NO: 88; amino acids 267-372 of SEQ ID NO: 89; amino acids 327-429 of SEQ ID NO: 90; amino acids 264-364 of SEQ ID NO: 91; amino acids 400-501 of SEQ ID NO: 92; amino acids 354-455 of SEQ ID NO: 93; amino acids 352-450 of SEQ ID NO: 94; amino acids 281-375 of SEQ ID NO: 95; amino acids 376-478 of SEQ ID NO: 96; amino acids 313-407 of SEQ ID NO: 97; amino acids 211-308 of SEQ ID NO: 98; amino acids 321-426 of SEQ ID NO: 99; amino acids 303-406 of SEQ ID NO: 100; amino acids 247-352 of SEQ ID NO: 101; amino acids 237-352 of SEQ ID NO: 101; amino acids 247-350 of SEQ ID NO: 102; amino acids 262-366 of SEQ ID NO: 103; or amino acids 233-366 of SEQ ID NO: 103.

Example 5 Construction of a Multivalent Clostridial Neurotoxin Comprising a Binding Domain

A polynucleotide molecule encoding BoNT/A-Noci is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-Noci is a BoNT/A modified to replace amino acids 874-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with a nociceptin-RK targeting domain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-Noci can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A polynucleotide molecule encoding BoNT/A-NtermNoci is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-NtermNoci is a BoNT/A modified to replace amino acids in the second binding domain (the N-terminal binding region of the multivalent Clostridial toxin described in Example 1) corresponding to amino acids 874-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with SEQ ID NO: 152, a nociceptin-RK binding domain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-NtermNoci can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructs for multivalent Clostridial toxin BoNT/B-Noci, a modified BoNT/B where amino acids 861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 152; multivalent Clostridial toxin BoNT/C1-Noci, a modified BoNT/C1 where amino acids 869-1291 of SEQ ID NO: 3 are replaced with SEQ ID NO: 152; BoNT/D-Noci, multivalent Clostridial toxin BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 are replaced with SEQ ID NO: 152; BoNT/E-Noci, multivalent Clostridial toxin BoNT/E where amino acids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 152; BoNT/F-Noci, multivalent Clostridial toxin BoNT/F where amino acids 867-1274 of SEQ ID NO: 6 are replaced with SEQ ID NO: 152; BoNT/G-Noci, multivalent Clostridial toxin BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replaced with SEQ ID NO: 152; multivalent Clostridial toxin TeNT-Noci, TeNT where amino acids 882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 152; multivalent Clostridial toxin BaNT-Noci, BaNT where amino acids 858-1268 of SEQ ID NO: 9 are replaced with SEQ ID NO: 152; multivalent Clostridial toxin BuNT-Noci, BuNT where amino acids 848-1251 of SEQ ID NO: 10 are replaced with SEQ ID NO: 152.

Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a binding domain comprising, e.g., SEQ ID NO: 104; SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, or SEQ ID NO: 253.

Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a binding domain comprising, e.g., amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113; amino acids 20-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; amino acids 26-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; amino acids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210 or SEQ ID NO: 214; or amino acids 51-58 of SEQ ID NO: 200; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermGRPP, a pUCBHB1/BoNT/A-Noci construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding the BoNT/A-NtermNoci; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/BoNT/A-NtermNoci. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding the multivalent Clostridial toxin BoNT/A-NtermNoci operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding multivalent Clostridial toxins BoNT/B-Noci, BoNT/C1-Noci, BoNT/D-Noci, BoNT/E-Noci, BoNT/F-Noci, BoNT/G-Noci, TeNT-Noci, BaNT-Noci, BuNT-Noci, as well as multivalent Clostridial toxin indicated above comprising, e.g., SEQ ID NO: 104; SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113; amino acids 20-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; amino acids 26-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; amino acids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210 or SEQ ID NO: 214; or amino acids 51-58 of SEQ ID NO: 200; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234

Example 6 Construction of a Multivalent Clostridial Neurotoxin Comprising Three CCK-A Binding Sites

The following example illustrates how to make a retargeted multivalent Clostridial toxin comprising three CCK-A binding sites.

A polynucleotide molecule is constructed as described in Example 1 above, encoding BoNT/A with the native binding site replaced with 10 repeats of the CCK 58 amino acid sequence (see U.S. Pat. No. 6,843,998, hereby incorporated by reference herein in its entirety) and with the same CCK 58 sequence repeated twice at the N-terminus of the single chain. The two N-terminal repeats are constructed to be separated by a short amino acid region comprising the loop region of BoNT/A, and two cysteine residues spanning the protease sensitive site. The multivalent Clostridial toxin is termed multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2). The basic architecture of the amino acid construct is given in FIG. 2A.

If desired, an expression-optimized polynucleotide molecule encoding multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2) can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructs for C(CCK)N(CCKx2) multivalent Clostridial toxin's in which the translocation and endopeptidase domains are independently selected from BoNT/B, C1, D, E, F, G, or TeNT.

To construct pET29/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2), a pUCBHB1/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2) construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2); and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2). The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as Kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2), operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding any combination of translocation and endopeptidase domains derived from BoNT/A, B, C1, D, E, F, G or TeNT with CCK 58 domains similar positioned as in multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2).

Example 7 Purification and Quantification of Modified Clostridial Toxins

The following example illustrates methods useful for purification and quantification of any modified Clostridial toxins disclosed in the present specification.

For immobilized metal affinity chromatography (IMAC) protein purification, E. coli BL21 (DE3) cell pellets used to express a modified Clostridial toxin, as described in Example 7, are resuspended in Column Binding Buffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2x Protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, San Diego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen, Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol), and then are transferred to a cold Oakridge centrifuge tube. The cell suspension is sonicated on ice (10-12 pulses of 10 seconds at 40% amplitude with 60 seconds cooling intervals on a Branson Digital Sonifier) in order to lyse the cells and then is centrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate. An immobilized metal affinity chromatography column is prepared using a 20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.) packed with 2.5-5.0 mL of TALON™ SuperFlow Co²⁺ affinity resin (BD Biosciences-Clontech, Palo Alto, Calif.), which is then equilibrated by rinsing with 5 column volumes of deionized, distilled water, followed by 5 column volumes of Column Binding Buffer. The clarified lysate is applied slowly to the equilibrated column by gravity flow (approximately 0.25-0.3 mL/minute). The column is then washed with 5 column volumes of Column Wash Buffer (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol). The modified Clostridial toxin is eluted with 20-30 mL of Column Elution Buffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and is collected in approximately twelve 1 mL fractions. The amount of modified Clostridial toxin contained in each elution fraction is determined by a Bradford dye assay. In this procedure, 20 μL aliquots of each 1.0 mL fraction is combined with 200 μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, Calif.), diluted 1 to 4 with deionized, distilled water, and then the intensity of the colorimetric signal is measured using a spectrophotometer. The five fractions with the strongest signal are considered the elution peak and are combined together. Total protein yield is determined by estimating the total protein concentration of the pooled peak elution fractions using bovine gamma globulin as a standard (Bio-Rad Laboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desalting column, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences, Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer (50 mM sodium phosphate, pH 6.5). After the column is equilibrated, a modified Clostridial toxin sample is applied to the size exclusion column with an isocratic mobile phase of 4° C. Column Buffer and at a flow rate of 10 mL/minute using a BioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules, Calif.). The desalted modified Clostridial toxin sample is collected as a single fraction of approximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ion exchange column, a modified Clostridial toxin sample that has been desalted following elution from an IMAC column is applied to a 1 mL Q1™ anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) using a BioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules, Calif.). The sample is applied to the column in 4° C. Column Buffer (50 mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C. Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) as follows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1 mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1 mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0 mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0 mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0 mL/minute. Elution of modified Clostridial toxin from the column is monitored at 280, 260, and 214 nm, and peaks absorbing above a minimum threshold (0.01 au) at 280 nm are collected. Most of the modified Clostridial toxin will elute at a sodium chloride concentration of approximately 100 to 200 mM. Average total yields of modified Clostridial toxin will be determined by a Bradford assay.

Expression of a modified Clostridial toxin is analyzed by polyacrylamide gel electrophoresis. Samples purified using the procedure described above are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) and are separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories, Hercules, Calif.) and the separated polypeptides are imaged using a Fluor-S MAX Multilmager (Bio-Rad Laboratories, Hercules, Calif.) for quantification of modified Clostridial toxin expression levels. The size and amount of modified Clostridial toxin is determined by comparison to MagicMark™ protein molecular weight standards (Invitrogen, Inc, Carlsbad, Calif.).

Expression of modified Clostridial toxin is also analyzed by Western blot analysis. Protein samples purified using the procedure described above are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) and are separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. Separated polypeptides are transferred from the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SD semi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules, Calif.). PVDF membranes are blocked by incubating at room temperature for 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serum albumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C. for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-Buffered Saline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate) containing appropriate primary antibodies as a probe. Primary antibody probed blots are washed three times for 15 minutes each time in Tris-Buffered Saline TWEEN-20. Washed membranes are incubated at room temperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing an appropriate immunoglobulin G antibody conjugated to horseradish peroxidase as a secondary antibody. Secondary antibody-probed blots are washed three times for 15 minutes each time in Tris-Buffered Saline TWEEN-20®. Signal detection of the labeled modified Clostridial toxin are visualized using the ECL Plus™ Western Blot Detection System (Amersham Biosciences, Piscataway, N.J.) and are imaged with a Typhoon 9410 Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) for quantification of modified Clostridial toxin expression levels.

Example 8 Treatment of Hyperhidrosis Using a Multivalent Clostridial Toxin

A 32-year-old woman presents complaining with chronic and excessive perspiring under the arms and in the palm of the hands. Clinical examination reveals a high degree of sweating under the arms, and stains on clothing in the same area.

The patient is injected in the eccrine glands under one arm with an approximately minimum effective dose (15 drops) of BOTOX®. The same patient is injected in the eccrine glands under the other arm with 7 drops of the Multivalent Clostridial Neurotoxin Derivative of Example 1.

The patient is observed one week later. Examination reveals that excessive sweating under the arms has been deceased by 85-95% in both cases, despite the fact that the multivalent Clostridial toxin was administered at less than 50% of the BOTOX® dosage.

Example 9 Treatment of Acute Pancreatitis with a Multivalent Clostridial Toxin

A 55 year-old man with a history of alcoholism presents with nausea, loss of appetite and severe abdominal pain radiating to the back. Examination reveals that the patient suffers from acute pancreatitis with a Balthazar Score of Grade D (with fluid collection in a single pancreatic location). The acute and advancing pancreatic necrosis threatens the patient's life.

The patient is administered the multivalent Clostridial toxin of Example 3 in an effective dose by injection directly into the pancreatic acini. Within 48 hours, there is a halt in the progression of the patient's deterioration. Within two weeks the acute pain has been relieved and the patient is able to take oral nourishment.

Although aspects of the present invention have been described with reference to the disclosed embodiments, one skilled in the art will readily appreciate that the specific examples disclosed are only illustrative of these aspects and in no way limit the present invention. Various modifications can be made without departing from the spirit of the present invention.

Any and all patents, publications, patent applications, and nucleotide and/or amino acid sequences referred to by accession numbers cited in this specification are hereby incorporated by reference as part of this specification.

Each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present invention provided that the features included in such a combination are not mutually inconsistent.

These and other aspects of the present invention are set forth in the following claims. 

1. A multivalent Clostridial toxin comprising: a) a Clostridial toxin enzymatic domain capable of executing an enzymatic target modification step of a Clostridial toxin intoxication process; b) a Clostridial toxin translocation domain capable of executing a translocation step of a Clostridial toxin intoxication process; c) a first binding domain capable of executing a cell binding step of a Clostridial toxin intoxication process by selectively binding a first cell surface receptor displayed by the target cell; d) a second binding domain capable of executing a cell binding step of a Clostridial toxin intoxication process by selectively binding a second cell surface receptor displayed by the target cell; and e) a protease cleavage site, wherein cleavage of the protease cleavage site converts the single-chain form of the modified Clostridial toxin into the di-chain form.
 2. The multivalent Clostridial toxin according to claim 1, wherein the multivalent Clostridial toxin comprises amino to carboxyl linear organization comprising a) a first binding domain, a translocation domain, a second binding domain, a protease cleavage site and an enzymatic domain, b) an enzymatic domain, a protease cleavage site, a first binding domain, a translocation domain and a second binding domain, c) an enzymatic domain, a protease cleavage site, a translocation domain, a first binding domain and a second binding domain, d) an enzymatic domain, a protease cleavage site, a translocation domain, a second binding domain and a first binding domain, e) a first binding domain, an enzymatic domain, a protease cleavage site, a translocation domain and a second binding domain, f) a translocation domain, a second binding domain, a protease cleavage site, a first binding domain and an enzymatic domain, g) a translocation domain, a second binding domain, a protease cleavage site, an enzymatic domain and a first binding domain, or h) a second binding domain, a translocation domain, a protease cleavage site, a first binding domain and an enzymatic domain.
 3. The multivalent Clostridial toxin according to claim 1, wherein the protease cleavage site is an endogenous protease cleavage site or an exogenous protease cleavage site.
 4. The multivalent Clostridial toxin according to claim 3, wherein the endogenous protease cleavage site is selected from the group consisting of a BoNT/A di-chain loop protease cleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/G di-chain loop protease cleavage site and a TeNT di-chain loop protease cleavage site.
 5. The multivalent Clostridial toxin according to claim 3, wherein the exogenous protease cleavage site is selected from the group consisting of a bovine enterokinase protease cleavage site, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C protease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site, and a Factor Xa protease cleavage site.
 6. The multivalent Clostridial toxin according to claim 1, wherein the Clostridial toxin enzymatic domain is selected from the group consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain and a BuNT enzymatic domain.
 7. The multivalent Clostridial toxin according claim 1, wherein the Clostridial toxin translocation domain is selected from the group consisting of a BoNT/A translocation domain, a BoNT/B translocation domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a BoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT translocation domain, a BaNT translocation domain and a BuNT translocation domain.
 8. The multivalent Clostridial toxin according claim 1, wherein the first binding domain comprises a binding domain selected from the group consisting of a Clostridial toxin binding domain, a Clostridial non-toxin associated protein β-trefoil domain and an FGF β-trefoil domain.
 9. The multivalent Clostridial toxin according claim 1, wherein the first binding domain comprises a binding domain selected from the group consisting of a glucagon like hormone, a neurohormone, a neuroregulatory cytokine, a neurotrophin, a growth factor, and an axon guidance signaling molecule.
 10. The multivalent Clostridial toxin according claim 1, wherein the first binding domain comprises a binding domain selected from the group consisting of an opioid peptide, a melanocortin peptide, a galanin peptide, a granin peptide, a tachykinin peptide, a cholecystokinin peptide, a Neuropeptide Y related peptide, a kinin peptide, a protease activated receptor (PAR) peptide, a somatostatin peptide, a leukemia inhibitor factor peptide, and an interleukin-1 peptide.
 11. The multivalent Clostridial toxin according claim 1, wherein the first binding domain comprises a protein translocation domain.
 12. The multivalent Clostridial toxin according to claim 11, wherein the PTD is selected from the group consisting of a herpes simplex virus type 1 VP22 protein translocating sequence, a SV-40 virus large T translocating sequence, a TAT translocating sequence, an adenovirus translocating sequence, a synthetic integrin binding domain translocating sequence, a Kaposi fibroblast growth factor membrane translocating sequence, a nuclear localization signal, a Transportan translocating sequence, a ciliary neurotrophic factor translocating sequence, a caveolin, an interleukin 1-β translocating sequence, a thioredoxin translocating sequence, a fibroblast growth factor-1 translocating sequence, a fibroblast growth factor-2 translocating sequence, an integrin β1 translocating sequence, an integrin β3 translocating sequence, a lactoferrin translocating sequence, a homeodomain translocating sequence, like, a penetratin translocating sequence, an Engrailed-1 translocating sequence, an Engrailed-2 translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4 translocating sequence, and a Hoxc-8 translocating sequence. 